High throughput generation and screening of fully human antibody repertoire in yeast

ABSTRACT

Compostions, kits and methods are provided for generating highly diverse libraries of proteins such as antibodies via homologous recombination in vivo, and screening these libraries against protein, peptide and nucleic acid targets using a two-hybrid method in yeast. The method for screening a library of tester proteins against a target protein or peptide comprises: expressing a library of tester proteins in yeast cells, each tester protein being a fusion protein comprised of a first polypeptide subunit whose sequence varies within the library, a second polypeptide subunit whose sequence varies within the library independently of the first polypeptide, and a linker peptide which links the first and second polypeptide subunits; expressing one or more target fusion proteins in the yeast cells expressing the tester proteins, each of the target fusion proteins comprising a target peptide or protein; and selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the tester fusion protein to the target fusion protein.

CROSS REFERENCE

This application is a continuation of U.S. Application No. 10,112,612,filed Mar. 27, 2002 which is a divisional of U.S. application Ser. No.09/603,663, filed Jun. 23, 2000, now U.S. Pat. No. 6,406,863. The aboveapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compositions, methods and kits for generatinglibraries of recombinant expression vectors and using these libraries inscreening of affinity-binding pairs, and, more particularly, forgenerating libraries of recombinant human antibodies and screening fortheir affinity binding with target antigens.

2. Description of Related Art

Antibodies are a diverse class of molecules. Delves, P. J. (1997)“Antibody production: essential techniques”, New York, John Wiley &Sons, pp. 90-113. It is estimated that even in the absence of antigenstimulation a human makes at least 10¹⁵ different antibody molecules—itsPermian antibody repertoire. The antigen-binding sites of manyantibodies can cross-react with a variety of related but differentantigenic determinants, and the Permian repertoire is apparently largeenough to ensure that there will be an antigen-binding site to fitalmost any potential antigenic determinant, albeit with low affinity.

Structurally, antibodies or immunoglobulins (Igs) are composed of one ormore Y-shaped units. For example, immunoglobulin G (IgG) has a molecularweight of 150 kDa and consists of just one of these units. Typically, anantibody can be proteolytically cleaved by the proteinase papain intotwo identical Fab (fragment antigen binding) fragments and one Fc(fragment crystallizable) fragment. Each Fab contains one binding sitefor antigen, and the Fc portion of the antibodies mediates other aspectsof the immune response.

A typical antibody contains four polypeptides-two identical copies of aheavy (H) chain and two copies of a light (L) chain, forming a generalformula H₂L₂. Each L chain is attached to one H chain by a disulfidebond. The two H chains are also attached to each other by disulfidebonds. Papain cleaves N-terminal to the disulfide bonds that hold the Hchains together. Each of the resulting Fabs consists of an entire Lchain plus the N-terminal half of an H chain; the Fc is composed of theC-terminal halves of two H chains. Pepsin cleaves at numerous sitesC-terminal to the inter-H disulfide bonds, resulting in the formation ofa divalent fragment [F(ab′)] and many small fragments of the Fc portion.IgG heavy chains contain one N-terminal variable (V_(H)) plus threeC-terminal constant (C_(H)1, C_(H)2 and C_(H)3) regions. Light chainscontain one N-terminal variable (V_(L)) and one C-terminal constant(C_(L)) region each. The different variable and constant regions ofeither heavy or light chains are of roughly equal length (about 110amino residues per region). Fabs consist of one V_(L), V_(H), C_(H)1,and C_(L) region each. The V_(L) and V_(H) portions containhypervariable segments (complementarity-determining regions or CDR) thatform the antibody combining site.

The V_(L) and V_(H) portions of a monoclonal antibody have also beenlinked by a synthetic linker to form a single chain protein (scFv) whichretains the same specificity and affinity for the antigen as themonoclonal antibody itself. Bird, R. E., et al. (1988) “Single-chainantigen-binding proteins” Science 242: 423-426. A typical scFv is arecombinant polypeptide composed of a V_(L) tethered to a V_(H) by adesigned peptide, such as (Gly₄-Ser)₃, that links the carboxyl terminusof the V_(L) to the amino terminus of the V_(H) sequence. Theconstruction of the DNA sequence encoding a scFv can be achieved byusing a universal primer encoding the (Gly₄-Ser)₃ linker by polymerasechain reactions (PCR). Lake, D. F., et al. (1995) “Generation of diversesingle-chain proteins using a universal (Gly₄-Ser)₃ encodingoligonucleotide” Biotechniques 19: 700-702.

The mammalian immune system has evolved unique genetic mechanisms thatenable it to generate an almost unlimited number of different light andheavy chains in a remarkably economical way by joining separate genesegments together before they are transcribed. For each type of Igchain—κ light chains, λ light chains, and heavy chain—there is aseparate pool of gene segments from which a single peptide chain iseventually synthesized. Each pool is on a different chromosome andusually contains a large number of gene segments encoding the V regionof an Ig chain and a smaller number of gene segments encoding the Cregion. During B cell development a complete coding sequence for each ofthe two Ig chains to be synthesized is assembled by site-specificgenetic recombination, bringing together the entire coding sequences fora V region and the coding sequence for a C region. In addition, the Vregion of a light chain is encoded by a DNA sequence assembled from twogene segments—a V gene segment and short joining or J gene segment. TheV region of a heavy chain is encoded by a DNA sequence assembled fromthree gene segments—a V gene segment, a J gene segment and a diversityor D segment.

The large number of inherited V, J and D gene segments available forencoding Ig chains makes a substantial contribution on its own toantibody diversity, but the combinatorial joining of these segmentsgreatly increases this contribution. Further, imprecise joining of genesegments and somatic mutations introduced during the V-D-J segmentjoining at the pre-B cell stage greatly increases the diversity of the Vregions.

After immunization against an antigen, a mammal goes through a processknown as affinity maturation to produce antibodies with higher affinitytoward the antigen. Such antigen-driven somatic hypermutation fine-tunesantibody responses to a given antigen, presumably due to theaccumulation of point mutations specifically in both heavy- andlight-chain V region coding sequences and a selected expansion ofhigh-affinity antibody-bearing B cell clones.

Great efforts have made to mimic such a natural maturation of antibodiesagainst various antigens, especially antigens associated with diseasessuch as autoimmune diseases, cancer, AIDS and asthma. In particular,phage display technology has been used extensively to generate largelibraries of antibody fragments by exploiting the capability ofbacteriophage to express and display biologically functional proteinmolecule on the its surface. Combinatorial libraries of antibodies havebeen generated in bacteriophage lambda expression systems which may bescreened as bacteriophage plaques or as colonies of lysogens (Huse etal. (1989) Science 246: 1275; Caton and Koprowski (1990) Proc. Natl.Acad. Sci. (U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad.Sci. (U.S.A.) 87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci.(U.S.A.) 88: 2432). Various embodiments of bacteriophage antibodydisplay libraries and lambda phage expression libraries have beendescribed (Kang et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363;Clackson et al. (1991) Nature 352: 624; McCafferty et al. (1990) Nature348: 552; Burton et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:10134; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133; Chang etal. (1991) J. Immunol. 147: 3610; Breitling et al. (1991) Gene 104: 147;Marks et al. (1991) J. Mol. Biol. 222: 581; Barbas et al. (1992) Proc.Natl. Acad. Sci. (U.S.A.) 89: 4457; Hawkins and Winter (1992) J.Immunol. 22: 867; Marks et al. (1992) Biotechnology 10: 779; Marks etal. (1992) J. Biol. Chem. 267: 16007; Lowman et al (1991) Biochemistry30: 10832; Lerner et al. (1992) Science 258: 1313). Also see review byRader, C. and Barbas, C. F. (1997) “Phage display of combinatorialantibody libraries” Curr. Opin. Biotechnol. 8: 503-508.

Various scFv libraries displayed on bacteriophage coat proteins havebeen described. Marks et al. (1992) Biotechnology 10: 779; Winter G andMilstein C (1991) Nature 349: 293; Clackson et al. (1991) op.cit.; Markset al. (1991) J. Mol. Biol. 222: 581; Chaudhary et al. (1990) Proc.Natl. Acad. Sci. (USA) 87: 1066; Chiswell et al. (1992) TIBTECH 10: 80;and Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879.

Generally, a phage library is created by inserting a library of a randomoligonucleotide or a cDNA library encoding antibody fragment such asV_(L) and V_(H) into gene 3 of M13 or fd phage. Each inserted gene isexpressed at the N-terminal of the gene 3 product, a minor coat proteinof the phage. As a result, peptide libraries that contain diversepeptides can be constructed. The phage library is then affinity screenedagainst immobilized target molecule of interest, such as an antigen, andspecifically bound phages are recovered and amplified by infection intoEscherichia coli host cells. Typically, the target molecule of interestsuch as a receptor (e.g., polypeptide, carbohydrate, glycoprotein,nucleic acid) is immobilized by covalent linkage to a chromatographyresin to enrich for reactive phage by affinity chromatography) and/orlabeled for screen plaques or colony lifts. This procedure is calledbiopanning. Finally, amplified phages can be sequenced for deduction ofthe specific peptide sequences. During the inherent nature of phagedisplay, the antibodies displayed on the surface of the phage may notadopt its native conformation under such in vitro selection conditionsas in a mammalian system. In addition, bacteria do not readily process,assemble, or express/secrete functional antibodies.

Transgenic animals such as mice have been used to generate fully humanantibodies by using the XENOMOUSE™ technology developed by companiessuch as Abgenix, Inc., Fremont, Calif. and Medarex, Inc. Annandale, N.J.Strains of mice are engineered by suppressing mouse antibody geneexpression and functionally replacing it with human antibody geneexpression. This technology utilizes the natural power of the mouseimmune system in surveillance and affinity maturation to produce a broadrepertoire of high affinity antibodies. However, the breeding of suchstrains of transgenic mice and selection of high affinity antibodies cantake a long period of time. Further, the antigen against which the poolof the human antibody is selected has to be recognized by the mouse as aforeign antigen in order to mount immune response; antibodies against atarget antigen that does not have immunogenicity in a mouse may not beable selected by using this technology. In addition, there may be aregulatory issue regarding the use of transgenic animals, such astransgenic goats (developed by Genzyme Transgenics, Framingham, Mass.)and chickens (developed by Geneworks, Inc., Ann Arbor, Mich.), toproduce antibody, as well as safety issues concerning containment oftransgenic animals infected with recombinant viral vectors.

Antibodies and antibody fragments have also been produced in transgenicplants. Plants, such as corn plants (developed by Integrated ProteinTechnologies, St. Louis, Mo.), are transformed with vectors carryingantibody genes, which results in stable integration of these foreigngenes into the plant genome. In comparison, most microorganismstransformed with plasmids can lose the plasmids during a prolongedfermentation. Transgenenic plant may be used as a cheaper means toproduce antibody in large scales. However, due to the long growthcircles of plants screening for antibody with high binding affinitytoward a target antigen may not be efficient and feasible for highthroughput screening in plants.

SUMMARY OF THE INVENTION

The present invention compositions, methods and kits for efficientlygenerating and screening for protein-protein or protein DNA bindingpairs in vivo. The production and screening of the binding pairs can beadopted for high throughput screening in vivo.

In one aspect of the present invention, compositions are provided. Thesecompositions may be used for screening affinity binding pairs between atester protein and a target molecule including protein, peptide, DNA,RNA, and small molecules in vitro or in vivo.

In one embodiment, a library of yeast expression vectors is provided.The yeast expression vectors forming in the library comprise a firstnucleotide sequence encoding a first polypeptide subunit; a secondnucleotide sequence encoding a second polypeptide subunit; and a linkersequence encoding a linker peptide that links the first nucleotidesequence and the second nucleotide sequence. The first polypeptidesubunit, the second polypeptide subunit, and the linker polypeptide areexpressed as a single fusion protein. In addition, the first and secondnucleotide sequence each independently varies within the library ofexpression vectors.

According to the embodiment, the yeast expression vector may be a 2μplasmid vector, preferably a yeast-bacterial shuttle vector whichcontains a bacterial origin of replication.

In another embodiment, a library of expression vectors is provided. Theexpression vectors forming in the library comprise: a transcriptionsequence encoding an activation domain or a DNA binding domain of atranscription activator; a first nucleotide sequence encoding a firstpolypeptide subunit; a second nucleotide sequence encoding a secondpolypeptide subunit; and a linker sequence encoding a linker peptidethat links the first nucleotide sequence and the second nucleotidesequence. The activation domain or the DNA binding domain of thetranscription activator, the first polypeptide subunit, the secondpolypeptide subunit, and the linker polypeptide are expressed as asingle fusion protein. In addition, the first and second nucleotidesequences each independently varies within the library of expressionvectors.

According to this embodiment, the expression vector may be a bacterial,phage, yeast, mammalian and viral expression vector, preferably a yeastexpression vector, and more preferably a 2μ plasmid yeast expressionvector.

Also according to this embodiment, the transcription activator sequencemay be located 5′ relative to the first nucleotide sequence, the linkersequence, and the second nucleotide sequence. Alternatively, thetranscription activator sequence may be located 3′ relative to the firstnucleotide sequence, the linker sequence, and the second nucleotidesequence.

In yet another embodiment, a library of transformed yeast cells isprovided. The library of yeast cells comprises a library of yeastexpression vectors. The expression vectors in the library of transformedyeast cells comprise: a transcription sequence encoding an activationdomain or a DNA binding domain of a transcription activator; a firstnucleotide sequence encoding a first polypeptide subunit; a secondnucleotide sequence encoding a second polypeptide subunit; and a linkersequence encoding a linker peptide that links the first nucleotidesequence and the second nucleotide sequence. The activation domain orthe DNA binding domain of the transcription activator, the firstpolypeptide subunit, the second polypeptide subunit, and the linkerpolypeptide are expressed as a single fusion protein. In addition, thefirst and second nucleotide sequences each independently varies withinthe library of expression vectors.

According to this embodiment, the yeast cells may be diploid yeastcells. Alternatively, the yeast cells may be haploids such as the a anda strain of yeast haploid cells.

In another aspect of the present invention, methods are provided forgenerating a library of yeast expression vectors that may be used forscreening protein-protein or protein-DNA binding pairs.

In one embodiment, the method comprises: transforming into yeast cells alinearized yeast expression vector having a 5′- and 3′-terminus sequenceat the site of linearization and a library of insert nucleotidesequences that are linear and double-stranded. The insert sequencescomprise a first nucleotide sequence encoding a first polypeptidesubunit, a second nucleotide sequence encoding a second polypeptidesubunit, and a linker sequence encoding a linker peptide that links thefirst and second polypeptide subunits. Each of the insert sequences alsocomprises a 5′- and 3′-flanking sequence at the ends of the insertsequence. The 5′- and 3′-flanking sequence of the insert sequence aresufficiently homologous to the 5′- and 3′-terminus sequences of thelinearized yeast expression vector, respectively, to enable homologousrecombination to occur. The homologous recombination occurring betweenthe vector and the insert sequence results in inclusion of the insertsequence into the vector in the transformed yeast cells.

In this embodiment, the first polypeptide subunit, the secondpolypeptide subunit, and the linker polypeptide are expressed as asingle fusion protein. Also, the first and second nucleotide sequenceseach independently varies within the library of expression vectors.

According to the embodiment, the 5′- or 3′-flanking sequence of theinsert nucleotide sequence may be preferably between about 30-120 bp inlength, more preferably between about 40-90 bp in length, and mostpreferably between about 60-80 bp in length.

In another embodiment, a method is provided for generating a library ofyeast expression vectors. The method comprises:

-   -   a) transforming into yeast cells        -   i) a linearized yeast expression vector having a 5′- and            3′-terminus sequence at a first site of linearization, and        -   ii) a library of first insert nucleotide sequences that are            linear, double stranded, each of the first insert sequences            comprising a first nucleotide sequence encoding a first            polypeptide subunit, a 5′- and 3′-flanking sequence at the            ends of the first insert sequence which are sufficiently            homologous to the 5′- and 3′-terminus sequences of the            vector at the first site of linearization, respectively, to            enable homologous recombination to occur;    -   b) having homologous recombination occur between the vector and        the first insert sequence in the transformed yeast cells, such        that the first insert sequence is included in the vector;    -   c) isolating from the transformed yeast cells the vectors that        contain the library of the first insert sequences;    -   d) linearizing the vectors containing the library of the first        insert sequences to generate a 5′- and 3′-terminus sequence at a        second site of linearization;    -   e) transforming into the transformed yeast cells        -   i) the linearized yeast expression vectors in step d), and        -   ii) a library of second insert nucleotide sequences that are            linear, double stranded, each of the second insert sequences            comprising a second nucleotide sequence encoding a second            polypeptide subunit, a 5′- and 3′-flanking sequence at the            ends of the second insert sequence which are sufficiently            homologous to the 5′- and 3′-terminus sequences of the            vector at the second site of linearization, respectively, to            enable homologous recombination to occur; and    -   f) having homologous recombination occur between the linearized        yeast expression vector at the second linearization site and the        second insert sequences in the transformed yeast cells, such        that the second insert sequence is included in the vector and        the first and second nucleotide sequences are linked by a linker        sequence.

The expression vectors formed by this method express the firstpolypeptide subunit, the second polypeptide subunit, and the linkerpolypeptide as a single fusion protein. Also, the first and secondnucleotide sequences each independently varies within the library ofexpression vectors formed by this method.

According to the embodiment, the 5′- or 3′-flanking sequence of theinsert nucleotide sequence are preferably between about 30-120 bp inlength, more preferably between about 40-90 bp in length, and mostpreferably between about 60-80 bp in length.

In a variation of the above-described method, the diversity of thelibrary of expression vectors formed by this method may be increased bychain shuffling via site-specific recombination. Accordingly, the methodmay further comprise: causing site-specific recombination between themembers of the library of the yeast expression vectors at the 5′- and3′-recombination sites, the recombination resulting in exchange of thefirst or second nucleotide sequences between the members of the libraryof the yeast expression vectors.

According to this variation, the 5′- and 3′-flanking sequences at theends of the first or second insert nucleotide sequence comprise a 5′-and 3′-recombination site, respectively, that are recognized by asite-specific recombinase.

Also according to the variation, the 5′- and 3′-site-specificrecombination sites are preferably different site-specific recombinationsites, more preferably sites which are each independently selected fromthe group consisting of SEQ ID Nos: 1-13, most preferably loxP ofcoliphase P1, and the other being a mutant loxP sequence.

Also according to this variation, the site-specific recombinase may beconstitutively or inducibly expressed in the yeast cells. Thesite-specific recombinase may be CRE recombinase that cause thesite-specific recombination.

In yet another aspect of the present invention, methods are provided forselecting tester proteins capable of binding to a target peptide,protein, or DNA.

In one embodiment where the target molecule is a target peptide orprotein, the method comprise: expressing a library of tester proteins inyeast cells, each tester protein being a fusion protein comprised of afirst polypeptide subunit whose sequence varies within the library, asecond polypeptide subunit whose sequence varies within the libraryindependently of the first polypeptide, and a linker peptide which linksthe first and second polypeptide subunits; expressing one or more targetfusion proteins in the yeast cells expressing the tester proteins, eachof the target fusion proteins comprising a target peptide or protein;and selecting those yeast cells in which a reporter gene is expressed,the expression of the reporter gene being activated by binding of thetester fusion protein to the target fusion protein.

According to this embodiment, expression of the reporter gene may beactivated by a functional transcription activator being formed by thebinding of the tester protein to the target peptide or protein as in ayeast two-hybrid system.

According, in a variation of the embodiment involving the yeasttwo-hybrid system, the step of expressing the library of tester fusionproteins may include transforming a library of tester expression vectorsinto the yeast cells which contain a reporter construct comprising thereporter gene whose expression is under transcriptional control of atranscription activator comprising an activation domain and a DNAbinding domain. Each of the tester expression vectors comprises a firsttranscription sequence encoding either the activation domain or the DNAbinding domain of the transcription activator, a first nucleotidesequence encoding the first polypeptide subunit, a second nucleotidesequence encoding the second polypeptide subunit, and a linker sequenceencoding a linker peptide that links the first nucleotide sequence andthe second nucleotide sequence. Optionally, the step of expressing thetarget fusion proteins includes transforming a target expression vectorinto the yeast cells simultaneously or sequentially with the library oftester expression vectors. The target expression vector comprises asecond transcription sequence encoding either the activation domain orthe DNA binding domain of the transcription activator which is notexpressed by the library of tester expression vectors; and a targetsequence encoding the target protein or peptide.

In another variation of the embodiment involving the yeast two-hybridsystem, the steps of expressing the library of tester fusion proteinsand expressing the target fusion protein includes causing mating betweenfirst and second populations of haploid yeast cells of opposite matingtypes. The first population of haploid yeast cells comprises a libraryof tester expression vectors for the library of tester fusion proteins.Each of the tester expression vector comprises a first transcriptionsequence encoding either the activation domain or the DNA binding domainof the transcription activator, a first nucleotide sequence encoding thefirst polypeptide subunit, a second nucleotide sequence encoding thesecond polypeptide subunit, and a linker sequence encoding a linkerpeptide that links the first nucleotide sequence and the secondnucleotide sequence. The second population of haploid yeast cellscomprises a target expression vector. The target expression vectorcomprises a second transcription sequence encoding either the activationdomain or the DNA binding domain of the transcription activator which isnot expressed by the library of tester expression vectors; and a targetsequence encoding the target protein or peptide. Either the first orsecond population of haploid yeast cells comprises a reporter constructcomprising the reporter gene whose expression is under transcriptionalcontrol of the transcription activator.

In this variation, the haploid yeast cells of opposite mating types maypreferably be α and a type strains of yeast. The mating between thefirst and second populations of haploid yeast cells of α and a typestrains may be conducted in a rich nutritional culture medium.

Optionally, a plurality of target fusion protein may be expressed andscreened against the library of tester proteins at the same time.According to this variation, the steps of expressing the library oftester fusion proteins and expressing the plurality of the target fusionproteins include causing mating between first and second populations ofhaploid yeast cells of opposite mating types. The first population ofhaploid yeast cells comprises a library of tester expression vectors forthe library of tester fusion proteins. Each of the tester expressionvector comprises a first transcription sequence encoding either theactivation domain or the DNA binding domain of the transcriptionactivator, a first nucleotide sequence encoding the first polypeptidesubunit, a second nucleotide sequence encoding the second polypeptidesubunit, and a linker sequence encoding a linker peptide that links thefirst nucleotide sequence and the second nucleotide sequence. The secondpopulation of haploid yeast cells comprises a plurality of targetexpression vectors. The target expression vectors comprise a secondtranscription sequence encoding either the activation domain or the DNAbinding domain of the transcription activator which is not expressed bythe library of tester expression vectors; and a target sequence encodingthe target protein or peptide. Either the first or second population ofhaploid yeast cells comprises a reporter construct comprising thereporter gene whose expression is under transcriptional control of thetranscription activator.

According to this variation, the haploid yeast cells of opposite matingtypes may preferably be α and a type strains of yeast. The matingbetween the first and second populations of haploid yeast cells of α anda type strains may be conducted in a rich nutritional culture medium.

Also according to this variation, members of the library of testerexpression vectors may be arrayed as individual yeast clones in one ormore multiple-well plates.

Also according to this variation, the plurality of the target expressionvectors may be arrayed as individual yeast clones in one or moremultiple-well plates.

Also according to this variation, the mating may be based on clonalmating in which each yeast clone containing a members of the testerexpression vectors is mated individually with each of the plurality oftarget expression vectors.

Also according to this variation, the plurality of the target expressionvectors may be a library of expression vectors containing a collectionof human EST clones or a collection of domain structures.

According to any of the above-described methods for selectingprotein-protein binding pairs, the target fusion protein comprises anantigen associated with a disease state such as a tumor-surface antigen.Optionally, the target fusion protein may comprises a human growthfactor receptor such as epidermal growth factors, transferrin,insulin-like growth factor, transforming growth factors, interleukin-1,and interleukin-2.

In another embodiment, a method is provided for screening protein-DNAbinding pairs in a yeast one-hybrid system.

The method comprises: expressing a library of tester fusion proteins inyeast cells which contain a reporter construct comprising a reportergene whose expression is under a transcriptional control of a target DNAsequence; and selecting the yeast cells in which the reporter gene isexpressed, the expression of the reporter gene being activated bybinding of the tester fusion protein to the target DNA sequence. Each ofthe tester fusion proteins comprises an activation domain of atranscription activator, a first polypeptide subunit whose sequencevaries within the library, a second polypeptide subunit whose sequencevaries within the library independently of the first polypeptidesubunit, and a linker peptide that links the first polypeptide subunitto the second polypeptide subunit.

In a variation of the embodiment, the step of expressing the library oftester fusion proteins includes transforming into the yeast cells alibrary of tester expression vectors for the library of tester fusionproteins. Each of the tester expression vectors comprises atranscription sequence encoding the activation domain of thetranscription activator, a first nucleotide sequence encoding the firstpolypeptide subunit, a second nucleotide sequence encoding the secondpolypeptide subunit, and a linker sequence encoding a linker peptidethat links the first nucleotide sequence and the second nucleotidesequence.

In another variation of the embodiment, the step of expressing a libraryof tester fusion proteins in yeast cells includes causing mating betweena first and second populations of haploid yeast cells of opposite matingtypes. The first population of haploid yeast cells comprises a libraryof tester expression vectors for the library of tester fusion proteins,each tester expression vector comprising a transcription sequenceencoding the activation domain of the transcription activator, a firstnucleotide sequence encoding the first polypeptide subunit, a secondnucleotide sequence encoding the second polypeptide subunit, and alinker sequence encoding a linker peptide that links the firstnucleotide sequence and the second nucleotide sequence. The secondpopulation of haploid yeast cells comprises the reporter construct.

According to the variation, the haploid yeast cells of opposite matingtypes may preferably be α and a type strains of yeast. The matingbetween the first and second populations of haploid yeast cells of α anda type strains is preferably conducted in a rich nutritional culturemedium.

According to any of the above-described methods for selectingprotein-DNA binding pairs, the target DNA sequence in the reporterconstruct is preferably positioned in 2-6 tandem repeats 5′ relative tothe reporter gene. The target DNA sequence in the reporter construct ispreferably between about 15-75 bp in length and more preferably betweenabout 25-55 bp in length.

In yet another embodiment, a method is provided for screeningprotein-protein binding pairs in a yeast one-hybrid system. The methodcomprises: expressing a library of tester fusion proteins in yeast cellswhich contain a reporter construct comprising a reporter gene whoseexpression is under a transcriptional control of a specific DNA bindingsite; expressing a target protein in the yeast cells expressing thetester fusion proteins, where the target protein binds to the specificDNA binding site; and selecting the yeast cells in which the reportergene is expressed, the expression of the reporter gene being activatedby binding of the tester fusion protein to the target protein. Each ofthe tester fusion proteins comprises an activation domain of atranscription activator, a first polypeptide subunit, a secondpolypeptide subunit, and a linker peptide that links the firstpolypeptide subunit to the second polypeptide subunit, wherein thesequences of the first and second polypeptide subunits eachindependently varies within the library of the tester fusion protein.

In a variation of the embodiment, the step of expressing the library oftester fusion proteins includes transforming into the yeast cells alibrary of tester expression vectors for the library of tester fusionproteins. Each of the tester expression vectors comprises atranscription sequence encoding the activation domain of thetranscription activator, a first nucleotide sequence encoding the firstpolypeptide subunit, a second nucleotide sequence encoding the secondpolypeptide subunit, and a linker sequence encoding a linker peptidethat links the first nucleotide sequence and the second nucleotidesequence.

In another variation of the embodiment, the steps of expressing thelibrary of tester fusion proteins and expressing the target fusionprotein includes causing mating between a first and second populationsof haploid yeast cells of opposite mating types. The first population ofhaploid yeast cells comprises a library of tester expression vectors forthe library of tester fusion proteins. Each of the tester expressionvectors comprises a transcription sequence encoding the activationdomain of the transcription activator, a first nucleotide sequenceencoding the first polypeptide subunit, a second nucleotide sequenceencoding the second polypeptide subunit, and a linker sequence encodinga linker peptide that links the first nucleotide sequence and the secondnucleotide sequence. The second population of haploid yeast cellscomprises a target expression vector comprising a target sequenceencoding the target protein. Either the first or second population ofhaploid yeast cells comprises the reporter construct.

In any of the above-described methods for selecting tester proteinscapable of binding to a target peptide, protein, or DNA, the method mayfurther comprise isolating the tester expression vectors from theselected yeast cells; and mutagenizing the first and second nucleotidesequences in the isolated tester expression vectors to form a library ofmutagenized expression vectors.

Examples of mutagenesis methods include, but are not limited to,error-prone PCR mutagenesis, site-directed mutagenesis, DNA shufflingand combinations thereof. The library of mutagenized expression vectorsmay be screened against the same or different target peptide, protein orDNA by following similar procedures used for screening the testerexpression vectors.

In yet another aspect of the present invention, methods are provided forproducing a library of single chain antibodies. In an embodiment, themethod comprises: expressing in yeast cells a library of yeastexpression vectors. Each of the yeast expression vector comprises afirst nucleotide sequence encoding an antibody heavy chain variableregion, a second nucleotide sequence encoding an antibody light chainvariable region, and a linker sequence encoding a linker peptide thatlinks the antibody heavy chain variable region and the antibody lightchain variable region. The antibody heavy chain variable region, theantibody light chain variable region, and the linker peptide areexpressed as a single fusion protein. Also, the first and secondnucleotide sequences each independently varies within the library ofexpression vectors to generate a library of single-chain antibodies witha diversity of at least 10⁶.

According to the embodiment, the diversity of the library ofsingle-chain antibodies is preferably between 10⁶-10¹⁶, more preferablybetween 10⁸-10¹⁶, and most preferably between 10¹⁰-10¹⁶.

In yet another aspect of the present invention, a kit is provided forselecting selecting tester proteins capable of binding to a targetpeptide, protein, or DNA.

In an embodiment, the kit comprises: a library of tester expressionvectors and a yeast cell line. Each of the tester expression vectorscomprises a first transcription sequence encoding either an activationdomain or a DNA binding domain of a transcription activator, a firstnucleotide sequence encoding a first polypeptide subunit, a secondnucleotide sequence encoding a second polypeptide subunit, and a linkersequence encoding a linker peptide that links the first nucleotidesequence and the second nucleotide sequence. The first and secondnucleotide sequences each independently varies within the library ofexpression vectors. A reporter construct may be contained in the yeastcell line. The reporter construct comprises a reporter gene whoseexpression is under a transcriptional control of a specific DNA bindingsite.

Optionally, the kit may further comprise a target expression vectorwhich comprises a second transcription sequence encoding either theactivation domain or the DNA binding domain of the transcriptionactivator which is not expressed by the library of tester expressionvectors; and a target sequence encoding the target protein or peptide.

In another embodiment, the kit comprises: a first and second populationsof haploid yeast cells of opposite mating types. The first population ofhaploid yeast cells comprises a library of tester expression vectors forthe library of tester fusion proteins. Each of the tester expressionvector comprises a first transcription sequence encoding either anactivation domain or a DNA binding domain of a transcription activator,a first nucleotide sequence encoding a first polypeptide subunit, asecond nucleotide sequence encoding a second polypeptide subunit, and alinker sequence encoding a linker peptide that links the firstnucleotide sequence and the second nucleotide sequence. The secondpopulation of haploid yeast cells comprises a target expression vector.The target expression vector encodes either the activation domain or theDNA binding domain of the transcription activator which is not expressedby the library of tester expression vectors; and a target sequenceencoding the target protein or peptide. Either the first or secondpopulation of haploid yeast cells comprises a reporter constructcomprising a reporter gene whose expression is under transcriptionalcontrol of the transcription activator.

Optionally, the second population of haploid yeast cells comprises aplurality of target expression vectors. Each of the target expressionvectors encodes either the activation domain or the DNA binding domainof the transcription activator which is not expressed by the library oftester expression vectors; and a target sequence encoding the targetprotein or peptide. Either the first or second population of haploidyeast cells comprises a reporter construct comprising a reporter genewhose expression is under transcriptional control of the transcriptionactivator.

According to any of the above-described compositions, methods and kits,the diversity of the first and/or the second polypeptide subunit encodedby the first and second nucleotide sequences within the library ofexpression vectors is preferably between 10³-10⁸, more preferablybetween 10⁴-10⁸, and most preferably between 10⁵-10⁸.

Also according to any of the above-described compositions, methods andkits, the diversity of the fusion proteins encoded by the library ofexpression vectors may be preferably at least 10⁶-10¹⁸, more preferablyat least 10⁹-10^(18,) and most preferably at least 10¹⁰-10¹⁸.

Also according to any of the above-described compositions, methods andkits, the diversities of the first and second polypeptide subunits maybe each independently derived from libraries of precursor sequences thatare not specifically designed for the target peptide or protein.

Also according to any of the above-described compositions, methods andkits, the diversities of the first and second polypeptide subunitsoptionally are not derived from one or more proteins that are known tobind to the target peptide or protein.

Also according to any of the above-described compositions, methods andkits, the diversities of the first and second polypeptide subunitsoptionally are not generated by mutagenizing one or more proteins thatare known to bind to the target peptide or protein.

Also according to any of the above-described compositions, methods andkits, the first and the second polypeptide subunits may be subunits of amultimeric protein whose sequence varies within a library of multimericproteins. Examples of multimeric proteins include, but are not limitedto, growth factor receptors, T cell receptors, cytokine receptors,tyrosine kinase-associated receptors, and MHC proteins.

Also according to any of the above-described compositions, methods andkits, the first nucleotide sequence may be 5′ relative to the secondnucleotide sequence. The first nucleotide sequence in the library ofexpression vectors comprises a coding sequence of an antibodyheavy-chain variable region, and the second nucleotide sequencecomprises a coding sequence of an antibody light-chain variable region.The source of the coding sequences of the antibody light-chain andheavy-chain variable regions may be from human, non-human primate, orrodent. Optionally, the source of the coding sequences of the antibodylight-chain and heavy-chain variable regions may be from one or morenon-immunized animals. Preferably, the source of the coding sequences ofthe antibody light-chain and heavy-chain variable regions may be fromhuman fetal spleen, lymph nodes or peripheral blood cells.

Also according to any of the above-described compositions, methods andkits, the linker peptides expressed by the library of expression vectorsmay provide a substantially conserved conformation between the first andsecond polypeptide subunits across the fusion proteins expressed by thelibrary of expression vectors. This may be achieved by having thesequence of the linker peptides be substantially conserved across thelibrary.

Also according to any of the above-described compositions, methods andkits, the conformation of the fusion protein having the first and secondpolypeptide subunits linked by the linker peptide may mimic aconformation of a single chain antibody. This may be achieved byselection of a linker peptide sequence comprising a Gly-Gly-Gly-Gly-Serpeptide in 3 or 4 tandem repeats.

Also according to any of the above-described compositions, methods andkits, the linker sequences in the library of expression vectors ispreferably between 30-120 bp in length, more preferably between 45-102bp in length, and most preferably between 45-63 bp in length. The linkersequences in the library of expression vectors may optionally comprise anucleotide sequence encoding an amino acid sequence ofGly-Gly-Gly-Gly-Ser in 3 or 4 tandem repeats.

Also according to any of the above-described compositions, methods andkits, each of the expression vectors may further comprise a sequenceencoding an affinity tag. Examples of affinity tags include, but are notlimited to, polyhistidine tags, polyarginine tags,glutathione-S-transferase, maltose binding protein, staphylococcalprotein A tag, and EE-epitope tags.

Also according to any of the above-described compositions, methods andkits, the transcription activator may be any transcription activatorhaving separable DNA-binding and transcriptional activation domains.Examples of transcription activators include, but are not limited to,GAL4, GCN4, and ADR1 transcription activators.

Also according to any of the above-described compositions, methods andkits, the reporter protein encoded by the reporter gene may be anyreporter gene, expression of which shows a distinct genotype orphenotype in a cell. Examples of such a reporter protein include, butare not limited to, β-galactosidase, α-galactosidase, luciferase,β-glucuronidase, chloramphenicol acetyl transferase, secreted embryonicalkaline phosphatase, green fluorescent protein, enhanced bluefluorescent protein, enhanced yellow fluorescent protein, and enhancedcyan fluorescent protein.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates a flow chart of a process that may be used in thepresent invention to screen for high affinity antibodies.

FIG. 2 illustrates an embodiment of a method for generating a library ofexpression vectors by sequentially inserting V1 and V2 fragments into alinearized expression vector via homologous recombination.

FIG. 3 illustrates an embodiment of a method for generating a library ofexpression vectors by inserting a single fragment comprising V1 and V2segments into a linearized expression vector via homologousrecombination.

FIG. 4A illustrates an embodiment of a method for increasing thecomplexity of a library of expression vectors via CRE/LoxP mediatedrecombination.

FIG. 4B illustrates a variation of the method illustrated in FIG. 4Awhere different nutritional markers are included in two libraries ofexpression vectors.

FIG. 5 illustrates an embodiment of a method or selectingprotein-protein binding pair in a two-hybrid system where the expressionvectors carrying the AD and BD domains are co-transformed into yeast.

FIG. 6 illustrates an embodiment of the method for selectingprotein-protein binding pairs in a two-hybrid system where theexpression vectors carrying the AD and BD domains are introduced intodiploid yeast cells via mating between two haploid yeast strains.

FIG. 7 illustrates an embodiment of a method for selecting protein-DNAbinding pair in a one-hybrid system where the expression vector carryingthe AD domain is transformed into yeast.

FIG. 8 illustrates an embodiment of the method for selectingprotein-protein binding pairs in a one-hybrid system where theexpression vector carrying the AD domain is transformed into yeast.

FIG. 9 illustrates an embodiment of a high throughput method forselecting protein-protein binding pairs in a two-hybrid system where thelibrary of the tester expression vectors and the library of expressionvector carrying the target expression vectors are each arrayed inmulti-well plates.

FIG. 10 illustrates an embodiment of a method used for mutagenesis andfurther screening of the clones selected from a primary screening of thetester proteins carried by the expression vector of the presentinvention.

FIG. 11 illustrates secondary structures of single-chain variablefragments (scFv), antibody fragments (Fab), and a fully-assembledantibody.

FIG. 12 illustrates examples of functional expression systems forantibody selected by using the method of the present invention.

FIG. 13 illustrates the plasmid map of pACT2 and a method of modifyingpACT2 in order to introduce a (G₄S)₄ linker into the plasmid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions, kits and efficientmethods for preparing extremely diverse libraries of tester proteins,and selecting from these libraries proteins with high affinity andspecificity toward a target protein, peptide or DNA in vivo. In oneparticular embodiment, highly diverse libraries of human antibodies canbe produced and screened against virtually any target antigen by usingthe compositions, kits and methods of the present invention.

The present invention provides a general method for screening thesediverse libraries of tester proteins against a single or a plurality oftarget proteins or peptides.

The method comprises: expressing a library of tester proteins in yeastcells, each tester protein being a fusion protein comprised of a firstpolypeptide subunit whose sequence varies within the library, a secondpolypeptide subunit whose sequence varies within the libraryindependently of the first polypeptide, and a linker peptide which linksthe first and second polypeptide subunits; expressing one or more targetfusion proteins in the yeast cells expressing the tester proteins, eachof the target fusion proteins comprising a target peptide or protein;and selecting those yeast cells in which a reporter gene is expressed,the expression of the reporter gene being activated by binding of thetester fusion protein to the target fusion protein.

The library of tester proteins may be any multimeric proteins whereinthe first and second polypeptide subunit are subunits of a multimericprotein whose sequence varies within the library of tester proteins.

In a preferred embodiment, the library of tester proteins is a libraryof antibodies where the first and second polypeptide subunits are anantibody heavy-chain variable region and an antibody light-chainvariable region, respectively. The source of the coding sequences of theantibody light-chain and heavy-chain variable regions may be fromhumans, non-human primates, or rodents.

From these libraries of antibodies, antibodies with high affinity andspecificity are selected by screening against the libraries single or aplurality of target antigens and antibodies, in particular, in yeast.Compared to conventional approaches of generating monoclonal antibody byhybridoma technology and the recently developed XENOMOUSE® technology,the present invention provides a more efficient and economical way toscreen for fully human antibodies in a much shorter period of time. Moreimportantly, the production and screening of the antibody libraries canbe readily adopted for high throughput screening in vivo.

The library of tester proteins may be produced in vivo or in vitro byusing any methods known in the art. The present invention provides anovel method for generating and screening libraries of expressionvectors encoding these tester proteins against a single or a pluralityof target molecules in vivo. These methods are developed by exploitingthe intrinsic property of yeast—homologous recombination at an extremelyhigh level of efficiency.

FIG. 1 shows a flow chart delineating a preferred embodiment of theabove method of the present invention for generating and screeninghighly diverse libraries of single-chain human antibodies (scFv) inyeast. As illustrated in FIG. 1, a highly complex library of scFv isconstructed in yeast cells. In particular, cDNA libraries of the heavyand light chain variable regions (V_(H) and V_(L)) are transferred intoa yeast expression vector by direct homologous recombination between thesequences encoding V_(H) and V_(L), and the yeast expression vectorcontaining homologous recombination sites. The resulting expressionvector is called scFv expression vector. This primary antibody librarymay reach a diversity preferably between 10⁶-10¹², more preferablybetween 10⁷-10¹², and most preferably between 10⁸-10¹².

The complexity of the primary antibody library generated in yeast can befurther increased by “chain-shuffling” between the light or heavy chainsequences contained in the scFv expression vector via site-specifichomologous recombination, such as CRE/loxP recombination. This antibodylibrary may reach a complexity of 10¹⁸ after mutagenesis of the scFvsequences in the primary antibody library by exchanging the V_(H) orV_(L) sequences between two scFv vectors. Hence, the diversity of theresulting antibody library may preferably be between 10⁹-10¹⁸, morepreferably between 10¹⁰-10¹⁸, and most preferably between 10¹²⁻¹⁸.

The highly complex primary antibody libraries can be used in a widevariety of applications. In particular, this library is used forscreening of fully human antibody against a wide variety of targets,such as a defined antigen or a library of antigens associated withdiseases.

The screening for antibody-antigen interaction may be convenientlycarried out in yeast by using a yeast two-hybrid method. For example, alibrary of scFv expression vectors are introduced into yeast cells.Expression of the scFv antibody library in the yeast cells produces alibrary of scFv fusion (tester) proteins, each fusion protein comprisinga scFv and an activation domain (AD) of a transcription activator. Theyeast cells are also modified to express a recombinant fusion proteincomprising a DNA-binding domain (BD) of the transcription activator anda target antigen. The yeast cells are also modified to express areporter gene whose expression is under the control of a specific DNAbinding site. Upon binding of the scFv antibody from the library to thetarget antigen, the AD is brought into close proximity of BD, therebycausing transcriptional activation of a reporter gene downstream from aspecific DNA binding site to which the BD binds. It is noted that thelibrary of scFv expression vectors may contain the BD domain while themodified yeast cells express a fusion protein comprising the AD domainand the target antigen.

These scFv expression vectors may be introduced to yeast cells byco-transformation of diploid yeast cells or by direct mating between twostrains of haploid yeast cells. For example, the scFv expression vectorscontaining libraries of V_(H) and V_(L) and an expression vectorcontaining the target antigen can be used to co-transform diploid yeastcells in a form of yeast plasmid or bacteria-yeast shuttle plasmid.Alternatively, two strains haploid yeast cells (e.g. α- and a-typestrains of yeast), each containing the scFv expression vector and thetarget antigen expression vector, respectively, are mated to produce adiploid yeast cell containing both expression vectors. Preferably, thehaploid yeast strain containing the target antigen expression vectoralso contains the reporter gene positioned downstream of the specificDNA binding site.

The yeast clones containing scFv antibodies with binding affinity to thetarget antigen are selected based on phenotypes of the cells or otherselectable markers. The plasmids encoding these primary antibody leadscan be isolated and further characterized.

The sequences encoding V_(H) and V_(L) of the primary antibody leads aremutagenized in vitro to produce a secondary antibody library. The V_(H)and V_(L) sequences can be randomly mutagenized by “poison” PCR (orerror-prone PCR), by DNA shuffling, or by any other way of random orsite-directed mutagenesis (or cassette mutagenesis). After mutagenesisin the regions of V_(H) and V_(L), the complexity of the secondaryantibody library may reach 10⁴ or more. Overall, the combined diversityor complexity of the total antibody libraries generated by using themethods of the present invention, including the primary and thesecondary antibody libraries, may reach 10¹⁸ or more. The secondaryantibody library are further screened for antibodies that bind thetarget antigen at high affinity by using the yeast-2-hybrid method asdescribed above or other methods of screening in vivo or in vitro.

An advantage of the present invention is that the overall process ofgenerating, selecting and optimizing large, diverse libraries ofantibodies mimics the process of natural antibody diversification andmaturation in a mammal. In the natural process of antibody affinitymaturation, the affinity of the antibodies against their antigen(s) isprogressively increased with the passage of time after immunization,largely due to the accumulation of point mutations specifically in thecoding sequences of both the heavy- and light-chain variable regions.

According to the present invention, extensive diversification isachieved by recombination and mutagenesis of the V_(H) and V_(L) chainlibraries derived from a wide variety of sources including natural andartificial or synthetic sources. The homologous combination of V_(H) andV_(L) in vivo to form the primary library of single-chain antibodiesmimics the natural process of antibody gene assembly from differentpools of gene segments encoding V_(H) and V_(L) of the antibodies. Sincethe method is preferably practiced with yeast cells, the highlyefficient homologous recombination in yeast is particularly useful tofacilitate such assembly of V_(H) and V_(L) in vivo.

The fast proliferation rate of yeast cells and ease of handling makes aprocess of “molecular evolution” dramatically shorter than the naturalprocess of antibody affinity maturation in a mammal. Therefore, antibodyrepertoires with extremely high diversity can be produced and screeneddirectly in yeast cells at a much lower cost and higher efficiency thanprior processes such as the painstaking, stepwise “humanization” ofmonoclonal murine antibodies isolated by using the conventionalhybridoma technology (a “protein redesign”) or the recently-developedXENOMOUSE™ technology.

According to the “protein redesign” approach, murine monoclonalantibodies of desired antigen specificity are modified or “humanized” invitro in an attempt to reshape the murine antibody to resemble moreclosely its human counterpart while retaining the originalantigen-binding specificity. Riechmann et al. (1988) Nature 332:323-327. This humanization demands extensive, systematic geneticengineering of the murine antibody, which could take months, if notyears. Additionally, extensive modification of the backbone of themurine monoclonal antibody may result in reduced specificity andaffinity.

In comparison, by using the method of the present invention, fully humanantibodies with high affinity to a specified antigen or antigens can bescreened and isolated directly from yeast cells without going throughsite-by-site modification of the antibody, and without sacrifice ofspecificity and affinity of the selected antibodies.

The XENOMOUSE™ technology has been used to generate fully humanantibodies with high affinity by creating strains of transgenic micethat produce human antibodies while suppressing the endogenous murine Igheavy- and light-chain loci. However, the breeding of such strains oftransgenic mice and selection of high affinity antibodies can take along period of time. The antigen against which the pool of the humanantibody is selected has to be recognized by the mouse as a foreignantigen in order to mount immune response; antibodies against a targetantigen that does not have immunogenicity in a mouse may not be able tobe selected by using this technology.

In contrast, by using the method of the present invention, libraries ofantibody can not only be generated at a great diversity and complexityin yeast cells more efficiently and economically, but also be screenedagainst virtually any protein or peptide target regardless of itsimmunogenicity. According to the present invention, any protein/peptidetarget can be expressed as a fusion protein with a DNA-binding domain(or an activation domain) of a transcription activator and selectedagainst the library of antibody in a yeast-2-hybrid system. Moreover,multiple protein targets or a library of antigens may be arrayed inmultiple-well plates and screened against the library of antibodies in ahigh throughput and automated manner.

Also compared to other approaches using transgenic goats and chickens toproduce antibodies, the method of the present invention can be used toscreen and produce fully human antibodies in large amounts withoutinvolving serious regulatory issues regarding the use of transgenicanimals, as well as safety issues concerning containment of transgenicanimals infected with recombinant viral vectors.

By using the method of the present invention, many requisite steps inthe traditional construction of cDNA libraries can be eliminated. Forexample, the time-consuming and labor-intensive steps of ligation andrecloning of cDNA libraries into expression vectors can be eliminated bydirect recombination or “gap-filling” in yeast through generalhomologous recombination and/or site-specific recombination. Throughoutthe whole process of antibody library construction, the DNA fragmentsencoding V_(H) and V_(L) are directly incorporated into a linearizedyeast expression vector via homologous recombination without therecourse to extensive recloning.

Compared with the approach of using phage display to screen for highaffinity antibodies in vitro, the method of the present inventionprovides efficient ways of screening for high affinity antibodies ineukaryotic cells in vivo. By using phage display technology, human Igheavy- and light chain variable regions are cloned, combinatoriallyreasserted, expressed and displayed as antigen-binding human Fab or scFvfragements on the surface of filamentous phage. Winter et al. (1994)Ann. Rev. Immunol. 433-455; and Rader et al. (1997) Current Opinion inBiotechnol. 8: 503-508. The phage-displayed human antigen-bindingfragments are then screened for their ability to bind an immobilizedtarget antigen in vitro, a process called biopanning. When high affinityhuman antibodies are desired, the phage display approach can beproblematic, presumably due to non-native conformation of antibodydisplay on the surface and/or extensive selection or panning requiredfor selection under in vitro conditions which bear little resemblance tothe physiological condition of a human body. In contrast, by using themethod of the present invention antibodies are selected based on theirbinding affinity to the target antigen in vivo. The antibodies areexpressed in the cell, go through protein folding, and binds to itstarget antigen under a natural environment. Thus, the antibodiesselected by using the method of the present invention should be morefunctionally relevant than those selected by panning in vitro.

1. Libraries of the Expression Vectors of the Present Invention

The present invention provides a library of expression vectors. In oneembodiment, a library of yeast expression vectors are provided. Each ofthe yeast expression vectors in the library comprises a first nucleotidesequence V1 encoding a first polypeptide subunit; a second nucleotidesequence V2 encoding a second polypeptide subunit; and a linker sequenceL encoding a linker peptide that links the first nucleotide sequence andthe second nucleotide sequence. The first polypeptide subunit, thesecond polypeptide subunit, and the linker polypeptide are expressed asa single fusion protein. In addition, V1 and V2 each independentlyvaries within the library of expression vectors.

According to the embodiment, the yeast expression vector may be a 2μplasmid vector, preferably a yeast-bacterial shuttle vector whichcontains a bacterial origin of replication.

In a variation of the embodiment, V1 is a coding sequence of theheavy-chain variable region of an antibody V_(H). V2 is a codingsequence of the light-chain variable region of an antibody V_(L).

The linker sequence L may have a specific sequence, or may vary withinthe library of the yeast expression vectors. Where L varies within thelibrary, its sequence diversity preferably does not substantially alterthe resulting conformation of the fusion protein.

When V1 and V2 are expressed by the yeast expression vector in yeastcells, such as cells from the Saccharomyces cerevisiae strains, thefusion protein comprising the V1 and V2 polypeptide segments undergoes aprocess of protein folding to adopt one or more conformations. Thepeptide sequence encoded by the linker sequence L may facilitate thefolding by providing a flexible hinge between the V1 and V2 polypeptidesegments. The conformation(s) adopted by the fusion protein may havesuitable binding site(s) for a specific target protein. For example, thefusion protein may be a single-chain antibody scFv that binds to itsspecific target antigen.

In another embodiment, a library of expression vectors is provided. Theexpression vector in the library comprises: a transcription sequenceencoding an activation domain AD or a DNA binding domain BD of atranscription activator; a first nucleotide sequence V1 encoding a firstpolypeptide subunit; a second nucleotide sequence V2 encoding a secondpolypeptide subunit; and a linker sequence L encoding a linker peptidethat links the first nucleotide sequence and the second nucleotidesequence. The activation domain or the DNA binding domain of thetranscription activator, the first polypeptide subunit, the secondpolypeptide subunit, and the linker polypeptide are expressed as asingle fusion protein. In addition, V1 and V2 each independently varieswithin the library of expression vectors.

According to the embodiment, the expression vector may be anygene-transferring vector as long as it is able to introduce the libraryof expression vectors to a desired location within a host cell, such asby transformation, transfection and transduction of the expressionvector into a host cell. The expression vector may be a bacterial,phage, yeast, mammalian or a viral expression vector, preferably a yeastexpression vector, and more preferably a 2μ plasmid yeast expressionvector.

Also according to the embodiment, the transcription activator sequencemay be located 5′ relative to the first nucleotide sequence, the linkersequence, and the second nucleotide sequence. Alternatively, thetranscription activator sequence may be located 3′ relative to the firstnucleotide sequence, the linker sequence, and the second nucleotidesequence.

In a variation of the embodiment, V1 is a coding sequence of theheavy-chain variable region of an antibody V_(H). V2 is a codingsequence of the light-chain variable region of an antibody V_(L).Optionally, AD is an activation domain of yeast GAL 4 transcriptionactivator; and BD is a DNA binding domain of yeast GAL 4 transcriptionactivator.

The linker sequence L may have a specific sequence, or vary within thelibrary of the yeast expression vectors.

When V1 and V2 are expressed by the expression vector in host cells,such as cells from the Saccharomyces cerevisiae strains, the fusionprotein comprising the AD, V1- and V2-encoded polypeptide segmentsundergoes a process of protein folding to adopt one or moreconformations. The peptide sequence encoded by the linker sequence Lalso facilitates the folding by providing a flexible hinge between theV1- and V2-encoded polypeptide segments. The conformation(s) adopted bythe fusion protein of the AD, V1 and V2-encoded polypeptide segments mayhave suitable binding site(s) for a specific target protein. Forexample, the fusion protein of AD, V1- and V2-encoded polypeptidesegments may be a single-chain antibody scFv that binds to its specifictarget antigen. The AD domain of the fusion protein should be able toactivate transcription of gene(s) once the AD and BD domains arereconstituted to form an active transcription activator in vitro or invivo by a two-hybrid method.

According to any of the libraries described above, the diversity of thefirst and/or the second polypeptide subunit encoded by V1 and V2 withinthe library of expression vectors may be preferably between 10³-10⁸,more preferably between 10⁴-10⁸, and most preferably between 10⁵-10⁸.

According to any of the libraries described above, the diversity of thefirst and/or the second polypeptide subunit encoded by V1 and V2 withinthe library of expression vectors may be preferably at least 10³, morepreferably at least 10⁴, and most preferably at least 10⁵.

Also according to any of the libraries described above, the diversity ofthe fusion proteins encoded by the library of expression vectors ispreferably between 10⁶-10¹⁸, more preferably between 10⁹-10¹⁸, and mostpreferably between 10¹⁰-10¹⁸.

Also according to any of the libraries described above, the diversitiesof the first and second polypeptide subunits need not be derived frommutagenizing one or more proteins that are known to bind to a targetpeptide or protein. For example, the first and second polypeptidesubunits need not be derived from mutagenizing a single antibody (e.g.the antibody Herceptin®) which is known to bind to a target peptide orprotein (Her-2 receptor). This reflects a novel ability of the presentinvention to identify new protein-protein binding pairs from a randompool of sequences instead of having to know in advance a protein thatbinds to a target and then form a library of mutants from that knownbinding protein.

Also according to any of the libraries described above, the linkersequences L in the library of expression vectors is preferably between30-120 bp in length, more preferably between 45-102 bp in length, andmost preferably between 45-63 bp in length. The linker sequence in thelibrary of expression vectors preferably comprises a nucleotide sequenceencoding an amino acid sequence of Gly-Gly-Gly-Gly-Ser in 3 or 4 tandemrepeats. Also according to any of the libraries described above, thelinker peptides expressed by the library of expression vectorspreferably provide a substantially conserved conformation between thefirst and second polypeptide subunits across the fusion proteinsexpressed by the library of expression vectors. For example, a linkerpeptide Gly-Gly-Gly-Gly-Ser in 4 tandem repeats (G₄S)₄ [SEQ ID NO: 75]is believed to provide a substantially conserved conformation of scFvantibodies which preserves its antigen-binding site in the variableregions of the corresponding full antibody.

The elements of the expression vector in the library are described indetail below.

1) The Backbone of the Expression Vector

The expression vector of the present invention may be based on any typeof vector as long as the vector that can transform, transfect ortransduce a host cell. The expression vector contains a library of theV1 sequences and a library of V2 sequences, and preferably contains asequence encoding an activation domain (AD) of a transcriptionalactivator. The acceptor vector may be plasmids, phages or viral vectorsas long as it is able to replicate in vitro, or in a host cell, or toconvey the library of the V1 and V2 sequences to a desired locationwithin a host cell. Examples of host cells include, but are not limitedto, bacterial (e.g. E. coli, Bacillus subtilis, etc.), yeast, animal,plant, and insect cells.

In a preferred embodiment, the expression vector is based on a yeastplasmid, especially one from Saccharomyces cerevisiae. Aftertransformation of yeast cells, the exogenous DNA encoding the V1 and V2sequences are uptaken by the cells and subsequently expressed by thetransformed cells.

More preferably, the expression vector may be a yeast-bacteria shuttlevector which can be propagated in either Escherichia coli or yeastStruhl, et al. (1979) Proc. Natl. Acad. Sci. 76: 1035-1039. Theinclusion of E. coli plasmid DNA sequences, such as pBR322, facilitatesthe quantitative preparation of vector DNA in E. coli, and thus theefficient transformation of yeast.

The types of yeast plasmid vector that may serve as the shuttle may be areplicating vector or an integrating vector. A replicating vector isyeast vector that is capable of mediating its own maintenance,independent of the chromosomal DNA of yeast, by virtue of the presenceof a functional origin of DNA replication. An integrating vector reliesupon recombination with the chromosomal DNA to facilitate replicationand thus the continued maintenance of the recombinant DNA in the hostcell. A replicating vector may be a 2μ-based plasmid vector in which theorigin of DNA replication is derived from the endogenous 2μ plasmid ofyeast. Alternatively, the replicating vector may be an autonomouslyreplicating (ARS) vector, in which the “apparent” origin of replicationis derived from the chromosomal DNA of yeast. Optionally, thereplicating vector may be a centromeric (CEN) plasmid which carries inaddition to one of the above origins of DNA replication a sequence ofyeast chromosomal DNA known to harbor a centromere.

The vectors may be transformed into yeast cells in a closed circularform or in a linear form. Transformation of yeast by integratingvectors, although with inheritable stability, may not be efficient whenthe vector is in in a close circular form (e.g. 1-10 transformants perug of DNA). Linearized vectors, with free ends located in DNA sequenceshomologous with yeast chromosomal DNA, transforms yeast with higherefficiency (100-1000 fold) and the transforming DNA is generally foundintegrated in sequences homologous to the site of cleavage. Thus, bycleaving the vector DNA with a suitable restriction endonuclease, it ispossible to increase the efficiency of transformation and target thesite of chromosomal integration. Integrative transformation may beapplicable to the genetic modification of brewing yeast, providing thatthe efficiency of transformation is sufficiently high and the target DNAsequence for integration is within a region that does not disrupt genesessential to the metabolism of the host cell.

ARS plasmids, which have a high copy number (approximately 20-50 copiesper cell) (Hyman et al., 1982), tend to be the most unstable, and arelost at a frequency greater than 10% per generation. However, thestability of ARS plasmids can be enhanced by the attachment of acentromere; centromeric plasmids are present at 1 or 2 copies per celland are lost at only approximately 1% per generation.

The expression vector of the present invention is preferably based onthe 2μ plasmid. The 2μ plasmid is known to be nuclear in cellularlocation, but is inherited in a non-Mendelian fashion. Cells that lostthe 2μ plasmid have been shown to arise from haploid yeast populationshaving an average copy number of 50 copies of the 2μ plasmid per cell ata rate of between 0.001% and 0.01% of the cells per generation. Futcher& Cox (1983) J. Bacteriol. 154: 612. Analysis of different strains of S.cerevisiae has shown that the plasmid is present in most strains ofyeast including brewing yeast. The 2μ plasmid is ubiquitous andpossesses a high degree of inheritable stability in nature.

The 2μ plasmid harbors a unique bidirectional origin of DNA replicationwhich is an essential component of all 2μ-based vectors. The plasmidcontains four genes, REP1, REP2, REP3 and FLP which are required for thestable maintenance of high plasmid copy number per cell Jaysram et al.(1983) Cell 34: 95. The REP1 and REP2 genes encode trans-acting proteinswhich are believed to function in concert by interacting with the REP3locus to ensure the stable partitioning of the plasmid at cell division.In this respect, the REP3 gene behaves as a cis acting locus whicheffects the stable segregation of the plasmid, and is phenotypicallyanalogous to a chromosomal centromere. An important feature of the 2μplasmid is the presence of two inverted DNA sequence repeats (each 559base-pairs in length) which separate the circular molecule into twounique regions. Intramolecular recombination between the inverted repeatsequences results in the inversion of one unique region relative to theother and the production in vivo of a mixed population of two structuralisomers of the plasmid, designated A and B. Recombination between thetwo inverted repeats is mediated by the protein product of a gene calledthe FLP gene, and the FLP protein is capable of mediating high frequencyrecombination within the inverted repeat region. This site specificrecombination event is believed to provide a mechanism which ensures theamplification of plasmid copy number. Murray et al. (1987) EMBO J. 6:4205.

The expression vector may also contain an Escherichia coli origin ofreplication and E. coli antibiotic resistance genes for propagation andantibiotic selection in bacteria. Many E. coli origins are known,including ColE1, pMB1 and pBR322, The ColE origin of replication ispreferably used in this invention. Many E. coli drug resistance genesare known, including the ampicillin resistance gene, thechloramphenoicol resistance gene and the tetracycline resistance gene.In one particular embodiment, the ampicillin resistance gene is used inthe vector.

The transformants that carry the V1 and V2 sequences may be selected byusing various selection schemes. The selection is typically achieved byincorporating within the vector DNA a gene with a discernible phenotype.In the case of vectors used to transform laboratory yeast, prototrophicgenes, such as LEU2, URA3 or TRP1, are usually used to complementauxotrophic lesions in the host. However, in order to transform brewingyeast and other industrial yeasts, which are frequently polyploid and donot display auxotrophic requirements, it is necessary to utilize aselection system based upon a dominant selectable gene. In this respectreplicating transformants carrying 2μ-based plasmid vectors may beselected based on expression of marker genes which mediate resistanceto: antibiotics such as G418, hygromycin B and chloramphenicol, orotherwise toxic materials such as the herbicide sulfometuron methyl,compactin and copper.

2) The V1 and V2 Variable Sequences

The first and the second polypeptide subunits encoded by V1 and V2,respectively, may be subunits of any multimeric protein. The sequence ofthe multimeric protein varies within a library or a collection ofmultimeric proteins. Example of the multimeric proteins include, but arenot limited to antibodies, growth factor receptors, T cell receptors,cytokine receptors, tyrosine kinase-associated receptors, and MHCproteins.

In preferred embodiment, the multimeric proteins are a library ofantibodies, and more preferably human antibodies. For example, the firstand second polypeptide subunits encoded by the library of expressionvectors may be a human antibody heavy-chain variable region V_(H) and ahuman antibody light-chain variable region V_(L).

DNA sequences encoding human antibody V_(H) and V_(L) segments may bepolynucleotide segments of at least 30 contiguous base pairssubstantially encoding genes of the immunoglobulin superfamily. A. F.Williams and A. N. Barclay (1989) “The Immunoglobulin Gene Superfamily”,in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H. Rabbifts, eds.,Academic Press: San Diego, Calif., pp. 361-387. The V_(H) and V_(L)genes are most frequently encoded by human, non-human primate, avian,porcine, bovine, ovine, goat, or rodent heavy chain and light chain genesequences.

The library of DNA sequences encoding human antibody V_(H) and V_(L)segments may be derived from a variety of sources. For example, mRNAencoding the human antibody V_(H) and V_(L) libraries may be extractedfrom cells or organs from immunized or non-immunized animals or humans.Preferably, organs such as human fetal spleen and lymph nodes may beused. Peripheral blood cells from non-immunized humans may also be used.The blood samples may be from an individual donor, from multiple donors,or from combined blood sources.

The human antibody V_(H)- and V_(L)-coding sequences may be derived andamplified by using sets of oligonucleotide primers to amplify the cDNAof human heavy and light chains variable domains by polymerase chainreaction (PCR). Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837. For example, blood sample may be from healthy volunteers andB-lymphocyte in the blood can be isolated. RNA can be prepared byfollowing standard procedures. Cathala et al. (1983) DNA 3:329. The cDNAcan be made from the isolated RNA by using reverse transcriptase.

Alternatively, the V_(H)- and V_(L)-coding sequences may be derived froman artificially rearranged immunoglobulin gene or genes. For example,immunoglobulin genes may be rearranged by joining of germ line Vsegments in vitro to J segments, and, in the case of V_(H) domains, Dsegments. The joining of the V, J and D segments may be facilitated byusing PCR primers which have a region of random or specific sequence tointroduce artificial sequence or diversity into the products.

The fusion protein formed by linking V_(H) and V_(L) polypeptides isalso referred as a single-chain antibody, scFv. A typical scFv comprisesa V_(H) domain and a V_(L) domain in polypeptide linkage, generallylinked via a spacer/linker peptide L. The linker peptide sequence L mayencode an appropriately designed linker peptide, such as(Gly-Gly-Gly-Gly-Ser)₄ [SEQ. ID NO: 75] or equivalent linker peptide(s).The linker bridges the C-terminus of the first V region and N-terminusof the second, ordered as either V_(H)-L-V_(L) or V_(L)-L-V_(H).

A scFv may comprise additional amino acid sequences at the amino- and/orcarboxy-termini. For example, a single-chain antibody may comprise atether segment for linking to the constant regions of a complete or fullantibody. A functional single-chain antibody generally contains asufficient portion of an immunoglobulin superfamily gene product so asto retain the property of binding to a specific target molecule,typically a receptor or antigen (epitope).

Optionally, the variable sequences V1 and V2 of the library ofexpression vectors may also be derived from multimeric proteins otherthan antibodies. V1 and V2 may be different subunits of a non-antibodymultimeric protein, such as membrance proteins and cell surfacesreceptor proteins, e.g. insulin receptor, MHC proteins (e.g. class I MHCand class II MHC protein), CD3 receptor, T cell receptors, cytokinereceptors such as interleukin-2 (IL-2) receptor which is made of α, β,and γ subunits, tyrosine-kinase-associated receptors such as Src, Yes,Fgr, Lck, Lyn, Hck, and Blk. The tyrosine-kinase-associated receptorscontain SH2 and SH3 domains which are held there partly by theirinteractions with transmembrane receptor proteins and partly bycovalently attached lipid chains. For example, V1 and V2 sequences maybe mutagenized sequences of the SH2 and SH3 domains of atyrosine-kinase-associated receptor such as Src, respectively, which areincorporated into the expression of vector of the present invention andscreened against various ligands for this receptor.

It is noted that V1 and V2 sequences may also be derived from librariesof different and diverse proteins which may be monomeric, and linked bythe linker sequence L.

A reflection of the power and versatility of the methods of the presentinvention is that the V1 and V2 sequences need not be based in any wayon a protein sequence known to bind to the target. Instead, V1 and V2may be from any source and may have a diversity that is entirelyindependent from the target, or one or more lead proteins known to bindto the target.

3) The Target Proteins and Peptides

The target fusion protein may comprise any target protein or peptidethat may be expressed or otherwise present in a host cell. The targetprotein may be a member of library of proteins or peptides, such as acollection of human ESTs, a total library of human ESTs, a collection ofdomain structures (e.g. Zn-finger protein domains), or a totally randompeptide library.

For example, the target protein or peptide may be a disease-associatedantigen, such as tumor surface antigen such as B-cell idiotypes, CD20 onmalignant B cells, CD33 on leukemic blasts, and HER2/neu on breastcancer. Antibody selected against these antigens can be used in a widevariety of therapeutic and diagnostic applications, such as treatment ofcancer by direct administration of the antibody itself or the antibodyconjugated with a radioisotope or cytotoxic drug, and in a combinationtherapy involving coadministration of the antibody with achemotherapeutic agent, or in conjunction with radiation therapy.

Alternatively, the target protein may be a growth factor receptor.Examples of the growth factor include, but are not limited to, epidermalgrowth factors (EGFs), transferrin, insulin-like growth factor,transforming growth factors (TGFs), interleukin-1, and interleukin-2.For example, high expression of EGF receptors have been found in a widevariety of human epithelial primary tumors. TGF-α have been found tomediate an autocrine stimulation pathway in cancer cells. Several murinemonoclonal antibody have been demonstrated to be able to bind EGFreceptors, block the binding of ligand to EGF receptors, and inhibitproliferation of a variety of human cancer cell lines in culture and inxenograft medels. Mendelsohn and Baselga (1995) Antibodies to'growthfactors and receptors, in Biologic Therapy of Cancer, 2^(nd) Ed., J BLippincott, Philadelphia, pp 607-623. Thus, fully human antibodiesselected against these growth factors by using the method of the presentinvention can be used to treat a variety of cancer.

The target protein may also be cell surface protein or receptorassociated with coronary artery disease such as platelet glycoproteinIib/IIIa receptor, autoimmune diseases such as CD4, CAMPATH-1 and lipidA region of the gram-negative bacterial lipopolysaccharide. Humanizedantibodies against CD4 has been tested in clinical trials in thetreatment of patients with mycosis fungoides, generalized postularpsoriasis, severe psorisis, and rheumatoid arthritis. Antibodies againstlipid A region of the gram-negative bacterial lipopolysaccharide havebeen tested clinically in the treatment of septic shock. Antibodiesagainst CAMPATH-1 has also been tested clinically in the treatment ofagainst refractory rheumatoid arthritis. Thus, fully human antibodiesselected against these growth factors by using the method of the presentinvention can be used to treat a variety of autoimmune diseases. Vaswaniet al. (1998) “Humanized antibodies as potential therapeutic drugs”Annals of Allergy, Asthma and Immunology 81: 105-115.

The target protein or peptide may also be proteins or peptidesassociated with human allergic diseases, such as those inflammatorymediator protein, e.g. Interleukin-1 (IL-1), tumor necrosis factor(TNF), leukotriene receptor and 5-lipoxygenase, and adhesion moleculessuch as V-CAM/VLA-4. In addition, IgE may also serve as the targetantigen because IgE plays pivotal role in type I immediatehypersensitive allergic reactions such as asthma. Studies have shownthat the level of total serum IgE tends to correlate with severity ofdiseases, especially in asthma. Burrows et al. (1989) “Association ofasthma with serum IgE levels and skin-test reactivity to allergens” NewEngl. L. Med. 320: 271-277. Thus, fully human antibodies selectedagainst IgE by using the method of the present invention may be used toreduce the level of IgE or block the binding of IgE to mast cells andbasophils in the treatment of allergic diseases without havingsubstantial impact on normal immune functions.

The target protein may also be a viral surface or core protein which mayserve as an antigen to trigger immune response of the host. Examples ofthese viral proteins include, but are not limited to, glycoproteins (orsurface antigens, e.g., GP120 and GP41) and capsid proteins (orstructural proteins, e.g., P24 protein); surface antigens or coreproteins of hepatitis A, B, C, D or E virus (e.g. small hepatitis Bsurface antigen (SHBsAg) of hepatitis B virus and the core proteins ofhepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein (G-protein)or the fusion protein (F-protein) of respiratory syncytial virus (RSV);surface and core proteins of herpes simplex virus HSV-1 and HSV-2 (e.g.,glycoprotein D from HSV-2).

The target protein may also be a mutated tumor suppressor gene that havelost its tumor-suppressing function and may render the cells moresusceptible to cancer. Tumor suppressor genes are genes that function toinhibit the cell growth and division cycles, thus preventing thedevelopment of neoplasia. Mutions in tumor suppressor genes cause thecell to ignore one or more of the components of the network ofinhibitory signals, overcoming the cell cycle check points and resultingin a higher rate of controlled cell growth—cancer. Examples of the tumorsuppressor genes include, but are not limited to, DPC-4, NF-1, NF-2, RB,p⁵³, WT1, BRCA1 and BRCA2.

DPC-4 is involved in pancreatic cancer and participates in a cytoplasmicpathway that inhibits cell division. NF-1 codes for a protein thatinhibits Ras, a cytoplasmic inhibitory protein. NF-1 is involved inneurofibroma and pheochromocytomas of the nervous system and myeloidleukemia. NF-2 encodes a nuclear protein that is involved in meningioma,schwanoma, and ependymoma of the nervous system. RB codes for the pRBprotein, a nuclear protein that is a major inhibitor of cell cycle. RBis involved in retinoblastoma as well as bone, bladder, small cell lungand breast cancer. P53 codes for p53 protein that regulates celldivision and can induce apoptosis. Mutation and/or inaction of p53 isfound in a wide ranges of cancers. WT1 is involved in Wilms tumor of thekidneys. BRCA1 is involved in breast and ovarian cancer, and BRCA2 isinvolved in breast cancer. Thus, fully human antibodies selected againsta mutated tumor suppressor gene product by using the method of thepresent invention can be used to block the interactions of the geneproduct with other proteins or biochemicals in the pathways of tumoronset and development.

2. Construction of the Library of Expression Vectors of the PresentInvention

The library of expression vectors described above can be constructedusing a variety of recombinant DNA techniques. The present inventionprovides novel and efficient methods of constructing these libraries ofexpression vectors with extreme diversity of V1 and V2 in vivo and invitro.

The methods of the present invention are provided by exploiting theinherent ability of yeast cells to facilitate homologous recombinationat an extremely high efficiency. The mechanism of homologousrecombination in yeast and its applications is briefly described below.

Yeast Saccharomyces cerevisiae has an inherited genetic machinery tocarry out efficient homologous recombination in the cell. This mechanismis believed to benefit the yeast cells for chromosome repair purpose andtraditionally also called gap repair or gap filling. By this mechanismof efficient gap filling, mutations can be introduced into specific lociof the yeast genome. For example, a vector carrying the mutant genecontains two sequence segments that are homologous to the 5′ and 3′ openreading frame (ORF) sequences of the gene that is intended to beinterrupted or mutated. The plasmid also contains a positive selectionmarker such as a nutritional enzyme allele, such as ura3, or anantibiotic resistant marker such as Geneticine (g418) that are flankedthe be two homologous segments. This plasmid is linearized andtransformed into the yeast cells. Through homologous recombinationbetween the plasmid and the yeast genome at the two homologousrecombination sites, a reciprocal exchange of the DNA content occursbetween the wild type gene in the yeast genome and the mutant gene(including the selection marker gene) that are flanked by the twohomologous sequence segments. By selecting for the positive nutritionalmarker, surviving yeast cells will loose the original wild type gene andwill adopt the mutant gene. Pearson BM, Hernando Y, and Schweizer M,(1998) Yeast 14: 391-399. This mechanism has also been used to makesystematic mutations in all 6,000 yeast genes or ORFs for functionalgenomics studies. Because the exchange is reciprocal, similar approachhas been used successfully for cloning yeast genomic fragments intoplasmid vector. Iwasaki T, Shirahige K, Yoshikawa H, and Ogasawara N,Gene 1991, 109 (1): 81-87.

By using homologous recombination in yeast, gene fragments or syntheticoligonucleotides can also be cloned into a plasmid vector without aligation step. In this application, a targeted gene fragment is usuallyobtained by PCR amplification (or by using the conventional restrictiondigestion out of an original cloning vector). Two short fragmentsequences that are homologous to the plasmid vector are added to the 5′and 3′ of the target gene fragment in the PCR amplification. This can beachieved by using a pair of PCR primers that incorporate the addedsequences. The plasmid vector typically includes a positive selectionmarker such as nutritional enzyme allele such as ura3, or an antibioticresistant marker such as geneticin (g418). The plasmid vector islinearized by a unique restriction cut in between the sequencehomologies that are shared with the PCR-amplified target, therebycreating an artificial gap at the cleavage site. The linearized plasmidvector and the target gene fragment flanked by sequences homologous tothe plasmid vector are co-transformed into a yeast host strain. Theyeast recognizes the two stretches of sequence homologies between thevector and target fragment, and facilitates a reciprocal exchange of DNAcontents through homologous recombination at the gap. As theconsequence, the target fragment is automatically inserted into thevector without ligation in vitro.

There are a few factors that may influence the efficiency of homologousrecombination in yeast. The efficiency of the gap repair is correlatedwith the length of the homologous sequences flanking both the linearizedvector and the targeted gene. Preferably, a minimum of 30 base pairs maybe required for the length of the homologous sequence, and 80 base pairsmay give a near-optimized result. Hua, S. B. et al. (1997) “Minimumlength of sequence homology required for in vitro cloning by homologousrecombination in yeast” Plasmid 38: 91-96. In addition, the reciprocalexchange between the vector and gene fragment is strictlysequence-dependent, i.e. not causing frame shift in this type ofcloning. Therefore, such a unique characteristic of the gap-repaircloning assures insertion of gene fragments with both high efficiencyand precision. The high efficiency makes it possible to clone two orthree targeted gene fragments simultaneously into the same vector in onetransformation attempt. Raymond K., Pownder T. A., and Sexson S. L.,(1999) Biotechniques 26: 134-141. The nature of precision sequenceconservation through homologous recombination makes it possible to clonetargeted genes in question into expression or fusion vectors for directfunction examinations. So far many functional or diagnostic applicationshave been reported using homologous recombination. El-Deiry W. W., etal., Nature Genetics1: 45-49,1992 (for p53), and Ishioka C., et al.,PNAS, 94: 2449-2453,1997 (for BRCA1 and APC).

A library of gene fragments may also be constructed in yeast by usinghomologous recombination. For example, a human brain cDNA library can beconstructed as a two-hybrid fusion library in vector pJG4-5. GuidoftiE., and Zervos A. S. (1999) “In vivo construction of cDNA library foruse in the yeast two-hybrid systems” Yeast 15: 715-720. It has beenreported that a total of 6,000 pairs of PCR primers were used foramplification of 6,000 known yeast ORFs for a study of total yeastgenomic protein interaction. Hudson, J. Jr, et al. (1997) Genome Res. 7:1169-1173. Uetz et al. conducted a comprehensive analysis ofprotein-protein interactions in Saccharomyces cerevisiae. Uetz et al.(2000) Nature 403: 623-627. The protein-protein interaction map of thebudding yeast was studied by using a comprehensive system to examinetwo-hybrid interactions in all possible combinations between the yeastproteins. Ito et al. (2000) Proc. Natl. Acad. Sci. USA. 97: 1143-1147.The genomic protein linkage map of Vaccinia virus was studied byMcCraith S., Holtzman T., Moss B., and Fields, S. (2000) Proc. Natl.Acad. Sci. USA 97: 4879-4884.

According to the present invention, the V1 and V2 sequences areintroduced into an expression vector by homologous recombinationperformed directly in yeast cells.

1) Cloning of V1 and V2 in Separate Fragments into an Expression VectorThrough Two Independent Events of Homologous Recombination in Yeast

In one embodiment of the method for generating the library of expressionvectors, the V1 and V2 sequences may be cloned into an expression vectorin vivo in two separate fragments through two independent events ofhomologous recombination in yeast.

The method comprises:

-   -   a) transforming into yeast cells i) a linearized yeast        expression vector having a 5′- and 3′-terminus sequence at a        first site of linearization; and ii) a library of first insert        nucleotide sequences that are linear, double stranded, each of        the first insert sequences comprising a first nucleotide        sequence V1 encoding a first polypeptide subunit, a 5′- and        3′-flanking sequence at the ends of the first insert sequence        which are sufficiently homologous to the 5′- and 3′-terminus        sequences of the vector at the first site of linearization,        respectively, to enable homologous recombination to occur;    -   b) having homologous recombination occur between the vector and        the first insert sequence in the transformed yeast cells, such        that the first insert sequence is included in the vector;    -   c) isolating from the transformed yeast cells the vectors that        contain the library of the first insert sequences;    -   d) linearizing the vectors containing the library of the first        insert sequences to generate a 5′- and 3′-terminus sequence at a        second site of linearization;    -   e) transforming into yeast cells        -   i) the linearized yeast expression vectors in step d), and        -   ii) a library of second insert nucleotide sequences that are            linear, double stranded, each of the second insert sequences            comprising a second nucleotide sequence V2 encoding a second            polypeptide subunit, a 5′- and 3′-flanking sequence at the            ends of the second insert sequence which are sufficiently            homologous to the 5′- and 3′-terminus sequences of the            vector at the second site of linearization, respectively, to            enable homologous recombination to occur; and    -   f) having homologous recombination occur between the linearized        yeast expression vector at the second linearization site and the        second insert sequences in the transformed yeast cells, such        that the second insert sequence is included in the vector and        the first and second nucleotide sequences are linked by a linker        sequence L.

In this embodiment, the expression vector expresses the firstpolypeptide subunit, the second polypeptide subunit, and the linkerpolypeptide as a single fusion protein. Also, the first and secondnucleotide sequences each independently varies within the library ofexpression vectors.

According to the embodiment, the 5′- or 3′-flanking sequence of theinsert nucleotide sequence is preferably between about 30-120 bp inlength, more preferably between about 40-90 bp in length, and mostpreferably between about 60-80 bp in length.

FIG. 2 illustrates an embodiment of this method according to the presentinvention. The coding sequences for V1 (e.g., V_(H)) and V2 (e.g.,V_(L)) are carried by separate PCR fragments and cloned into anexpression vector sequentially following two independent events ofhomologous recombination in yeast.

As illustrated in FIG. 2, the V1 fragment has a 5′ flanking sequence anda 3′ flanking sequence that are homologous to the 5′ and 3′ terminus ofa linearized expression vector, respectively. When the V1 fragment andthe linearized expression vector are introduced into a host cell, forexample, transformed into a yeast cell, the “gap” (the firstlinearization site) created by linearization of the expression vector isfilled by the V1 fragment insert through recombination of the homologoussequences at the 5′ and 3′ terminus of these two linear double-strandedDNA. Through this event of homologous recombination, a library ofcircular vectors carrying the variable sequence V1 is generated.

This library of circular vectors is then cleaved at a secondlinearization site, for example, a site downstream of V1. The V2fragment has a 5′ flanking sequence and a 3′ flanking sequence that arehomologous to the 5′ and 3′ terminus of the linearized expression vectorat the second linearization site. The V2 fragment and the linearizedexpression vector are transformed into a yeast cell. Through a secondevent of homologous recombination, the V2 fragment is inserted into thelinearized expression vector at the second linearization site. As aresult, a library of circular vectors carrying the variable sequences V1and V2 is generated.

Each flanking sequence added to the V1 and V2 coding sequence may bepreferably between about 30-120 bp in length, more preferably betweenabout 40-100 bp in length, and most preferably 60-80 bp in length.

The region between the V1 and V2 sequences, i.e. the linker sequence L,is preferably be 30-120 bp in length, more preferably 45-102 bp inlength, and more most preferably 45-63 bp in length. The linker sequencepreferably codes for an amino acid sequence of Gly-Gly-Gly-Gly-Ser (G₄S)in multiple tandem repeats, more preferably codes for (G₄S)₃₋₆ and mostpreferably codes for (G₄S)₃₋₄. Optionally, the linker sequence mayfurther include a site-specific homologous recombination site, such as aloxP site.

When the V1 and V2 coding sequences are inserted into an expressionvector containing an AD domain, it is preferred that the reading framesof the V1 and V2 fragments are conserved with upstream AD reading frame.

Depending on the cloning expression vector used, additional featuressuch as affinity tags and unique restriction enzyme recognition sitesmay be added to the expression for the convenience of detection andpurification of the inserted V1 and V2 sequences. Examples of affinitytags include, but are not limited to, a polyhistidine tract,polyarginine, glutathione-S-transferase (GST), maltose binding protein(MBP), a portion of staphylococcal protein A (SPA), and variousimmunoaffinity tags (e.g. protein A) and epitope tags such as thoserecognized by the EE (Glu-Glu) antipeptide antibodies.

In a preferred embodiment, the V1 and V2 sequences may be the codingsequences for a heavy-chain variable region V_(H) and a light-chainvariable region V_(L), respectively, which are derived from a humanantibody repertoire. To generate the V1 and V2 coding sequences from thehuman antibody repertoire, a complex human scFv cDNA gene pool maygenerated by using the methods known in the art. Sambrook, J., et al.(1989) Molecular Cloning: a laboratory manual. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; and Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology” John Wiley & Sons, NY.

Total RNA may be isolated from sources such as the white cells (mainly Bcells) contained in peripheral blood supplied by un-immunized humans, orfrom human fetal spleen and lymph nodes. First strand cDNA synthesis maybe synthesized performed by using methods known in the art, such asthose described by Marks et al. Marks et al. (1991) Eur. J. Immunol. 21:985-991.

Specifically, a mixture of heavy and light chain cDNA primer setsdesigned to anneal to the constant regions may be used for priming thesynthesis of cDNA of heavy chain and light chains (both kappa Vκ andlambda Vλ) antibody genes. Examples of how to generate the cDNA libraryof human V_(H) and V_(L) genes are illustrated in Example 1.

The coding sequences of human heavy and light chain genes may beamplified from the V_(H) and V_(L) cDNA library generated above by usingPCR primer sets used in combination to prime the heavy chain variableregion V_(H), and the light chain variable regions Vλ and Vκ. The eachof the PCR primers may include both a V_(H), Vλ or Vκ partial sequenceand a 5′ or 3′ flanking sequence for facilitating homologousrecombination between the V_(H) and V_(L) fragments and a cloningexpression vector. Examples of these primers are listed in Table 2.

2) Cloning of V1 and V2 that is Assembled in a Single Fragment into anExpression Vector Via Homologous Recombination in Yeast.

In another embodiment of the method for generating the library ofexpression vectors, the V1 and V2 sequences may be assembled into asingle DNA fragment in vitro by using, for example, a PCR method. Thesingle fragment comprising V1, V2 and L may then be cloned into anexpression vector via homologous recombination in yeast.

The method comprises: transforming into yeast cells a linearized yeastexpression vector having a 5′- and 3′-terminus sequence at the site oflinearization and a library of insert nucleotide sequences that arelinear and double-stranded. Each of the insert sequences comprises afirst nucleotide sequence V1 encoding a first polypeptide subunit, asecond nucleotide sequence V2 encoding a second polypeptide subunit, anda linker sequence L encoding a linker peptide that links the first andsecond polypeptide subunits. Each of the insert sequences also comprisesa 5′- and 3′-flanking sequence at the ends of the insert sequence. The5′- and 3′-flanking sequence of the insert sequence are sufficientlyhomologous to the 5′- and 3′-terminus sequences of the linearized yeastexpression vector, respectively, to enable homologous recombination tooccur. The homologous recombination occurring between the vector and theinsert sequence results in inclusion of the insert sequence into thevector in the transformed yeast cells.

In this embodiment, the first polypeptide subunit, the secondpolypeptide subunit, and the linker polypeptide are expressed as asingle fusion protein. Also, the first and second nucleotide sequenceseach independently varies within the library of expression vectors.

According to the embodiment, the 5′- or 3′-flanking sequence of theinsert nucleotide sequence is preferably between about 30-120 bp inlength, more preferably between about 40-90 bp in length, and mostpreferably between about 60-80 bp in length.

FIG. 3 illustrates an embodiment of this method according to the presentinvention. The coding sequences for V1 (e.g., V_(H)) and V2 (e.g.,V_(L)) are amplified by PCR to generate separate fragements which arethen PCR-assembled into a single PCR fragment carrying both V1 and V2sequences. This single PCR fragment is then cloned into an expressionvector through homologous recombination in one step. The detailedprocedures are described in Example 1.

As illustrated in FIG. 3, the V1 fragment has a flanking sequence at its3′ terminus that overlaps with a flanking sequence of the 5′ terminus ofthe V2 fragment. By using a method of overlapping PCR priming, the V1and V2 fragments are assembled into a single PCR fragment with a linkagesequence L in between, which is referred to as the V1-L-V2 fragment.This single PCR fragment has a 5′ flanking sequence and a 3′ flankingsequence that are homologous to the 5′ and 3′ terminus of a linearizedexpression vector at the cleavage site, respectively. When the V1-L-V2fragment and the linearized expression vector are introduced into a hostcell, for example, transformed into a yeast cell, the “gap” created bylinearization of the expression vector is filled by the V1-L-V2 fragmentinsert through recombination of the homologous sequences at the 5′ and3′ terminus of these two linear double-stranded DNA. Through thishomologous recombination, a library of circular vectors carrying thevariable sequences V1 and V2 is generated.

Each flanking sequence added to the 5′ and 3′-terminus of V1 and V2coding sequence is preferably between about 30-120 bp in length, morepreferably between about 40-100 bp in length, and most preferably 60-80bp in length.

The region between the V1 and V2 sequences, i.e. the linker sequence L,is preferably 30-120 bp in length, more preferably 45-102 bp in length,and more most preferably 45-63 bp in length. The linker sequencepreferably codes for an amino acid sequence of Gly-Gly-Gly-Gly-Ser (G₄S)in multiple tandem repeats, more preferably codes for (G₄S)₃₋₆ and mostpreferably codes for (G₄S)₃₋₄. Optionally, the linker sequence mayfurther include a site-specific homologous recombination site, such as aloxP site.

By using similar methods as described above, the variable sequences V1and V2 can be inserted into an expression vector containing anactivation domain (AD) or a DNA-binding domain (BD) of a transcriptionactivator. The AD or BD domain may be positioned upstream or downstreamof V1 and V2. It is preferred that the reading frames of the V1 and V2fragments are conserved with the AD or BD reading frame.

The expression vector containing an AD (or BD) domain may be any vectorengineered to carry the coding sequence of the AD domain. The expressionvector is preferably a yeast vector such as pGAD10 (Feiloter et al.(1994) “Construction of an improved host strain for two hybridscreening” Nucleic Acids Res. 22: 1502-1503), pACT2 (Harper et al (1993)“The p21 Cdk-interacting protein Cip1 is a protein inhibitor of G1cyclin-dependent kinase” Cell 75:805-816), and pGADT7 (“Matchmaker Gal4two hybrid system 3 and libraries user manual” (1999), ClontechPT3247-1, supplied by Clontech, Palo Alto, Calif.).

Optionally, the expression vector containing an AD (or BD) domain mayalso include another expression unit which is capable of expressing oneor more proteins other than the fusion proteins encoded by V1 and V2,such as the scFv antibodies. These proteins may be modifying enzymessuch as kinase, glycosylase, and enzymes that help to form the disulfidebonds present in a mature antibody structure. Expression of the enzymesshould facilitate or enhance posttranslational modifications that may berequired for full functions of the fusion proteins encoded by V1 and V2.

Expression of these proteins may be under the transcriptional control ofa constitutive promoter or an inducible promoter. One example of such anexpression vector is available from Clontech, pBridge® (catalog No.6184-1). The expression vector, pBridge®, contains one expression unitthat controls expression of a Gal 4 BD domain and another expressionunit that includes an inducible promoter Pmat25. Tirode, E. et al.(1997) J. Biol. Chem. 272: 22995-22999.

The linearized vector DNA may be mixed with equal or excess amount ofthe PCR insert fragment: either V1 (or V2) in a separate fragment or inthe single fragment comprising V1 and V2. The linearized vector DNA andthe PCR fragment are co-transformed into host cells, such as competentyeast cells. Recombinant clones may be selected based on survival ofcells in a nutritional selection medium or based on other phenotypicmarkers. Either the linearized vector or the insert DNA fragment alonemay be used as a control for determining the efficiency of recombinationand transformation.

Other homologous recombination systems may be used to generate thelibrary of expression vectors of the present invention. For example, therecombination between the library of V1 and V2 sequences and therecipient expression vector may be facilitated by site-specificrecombination.

The site-specific recombination employs a site-specific recombinase, aenzyme which catalyzes the exchange of DNA segments at specificrecombination sites. Site-specific recombinases present in some virusesand bacteria, and have been characterized to have both endonuclease andligase properties. These recombinases, along with associated proteins insome cases, recognize specific sequences of bases in DNA and exchangethe DNA segments flanking those segments. Landy, A. (1993) CurrentOpinion in Biotechnology 3: 699-707.

A typical site-specific recombinase is CRE recombinase. CRE is a 38-kDaproduct of the cre (cyclization recombination) gene of bacteriophage P1and is a site-specific DNA recombinase of the Int family. Sternberg, N.et al. (1986) J. Mol. Biol. 187: 197-212. CRE recognizes a 34-bp site onthe P1 genome called loxP (locus of X-over of P1) and efficientlycatalyzes reciprocal conservative DNA recombination between pairs ofloxP sites. The loxP site [SEQ ID NO: 1] consists of two 13-bp invertedrepeats flanking an 8-bp nonpalindromic core region. CRE-mediatedrecombination between two directly repeated loxP sites results inexcision of DNA between them as a covalently closed circle. Cre-mediatedrecombination between pairs of loxP sites in inverted orientation willresult in inversion of the intervening DNA rather than excision.Breaking and joining of DNA is confined to discrete positions within thecore region and proceeds on strand at a time by way of transientphophotyrosine DNA-protein linkage with the enzyme.

The CRE recombinase also recognizes a number of variant or mutant loxsites relative to the loxP sequence. Examples of these Cre recombinationsites include, but are not limited to, the loxB, loxL and loxR siteswhich are found in the E. coli chromosome. Hoess et al. (1986) NucleicAcid Res. 14: 2287-2300. Other variant lox sites include, but are notlimited to, loxB, loxL, loxR, loxP3, loxP23, loxΔ86, loxΔ117, loxP511[SEQ ID NO:2], and loxC2 [SEQ ID NO:3]. Table 1 lists examples of loxsites that may be used in the present invention, including wild-typeloxP sites LoxP WT [SEQ ID NO: 1] and loxP2 [SEQ ID NO: 5], and otherloxP variants with mutations in the 13-bp inverted repeats region and/orthe 8-bp nonpalindromic core region (underlined), loxP511 [SEQ ID NO:2], loxC2 [SEQ ID NO: 3], loxP1 [SEQ ID NO: 4], loxP3 [SEQ ID NO: 6],loxP4 [SEQ ID NO: 7], loxP5 [SEQ ID NO: 8], loxP6 [SEQ ID NO: 9], loxP7[SEQ ID NO: 10], loxP8 [SEQ ID NO: 11], loxP9 [SEQ ID NO: 12], andloxP10 [SEQ ID NO: 13].

Examples of the non-CRE recombinases include, but are not limited to,site-specific recombinases include: att sites recognized by the Intrecombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL,and attR), the FRT sites recognized by FLP recombinase of the 2piplasmid of Saccharomyces cerevisiae, the recombination sites recognizedby the resolvase family, and the recombination site recognized bytransposase of Bacillus thruingiensis.

Subsequent analysis may also be carried out to determine the efficiencyof homologous recombination that results in correct insertion of the V1and V2 sequences into the expression vector. For example, PCRamplification of the V1 and V2 inserts directly from the selected yeastclone may reveal how many clones are recombinant. Libraries with minimumof 90% recombinant clones are preferred. The same PCR amplification ofselected clones may also reveal the insert size. Although a smallfraction of the library may contain double or triple inserts, themajority (>90%) is preferably to have a single insert with the expectedsize.

To verify sequence diversity of the inserts in the selected clones, PCRamplification product with the correct size of insert may befingerprinted with frequent digesting restriction enzymes. From a gelelectrophoresis pattern, it may be determined whether the clonesanalyzed are of the same identity or of the distinct or diversifiedidentity. The PCR products may also be sequenced directly to reveal theidentity of inserts and the fidelity of the cloning procedure and toprove the independence and diversity of the clones.

In an embodiment where the V1 and V2 sequences are the coding sequencesfor a heavy-chain variable region V_(H) and a light-chain variableregion V_(L) derived from a human antibody repertoire, respectively,mouse scFv fragments may be generated from hybridoma cell lines ascontrols by following the same procedures described above. Examples ofhybridoma cell lines include, but are not limit to, anti-GFP antibodyproducing cell line (Clontech), anti-p53 antibodies producing cell lines(NeoMarker), and other hybridoma cell lines available from ATCC(Atlanta). The hybridoma cell line is subjected to the same proceduresdescribed above, i.e., RNA isolation, cDNA synthesis, PCR amplification,and homologous recombination into yeast. Other scFv antibody librariesmay also be generated from mouse fetal liver and fetal spleen using thesame principle.

The mouse scFV library generated can provide a direct control forexisting individual mouse monoclonal antibody with its cognate antigen.Most studies for antigen-antibody interaction have been performed withmouse antibodies. The mouse scFV library should serve as an excellentcontrol in the selection of human scFv antibody library against a targetantigen by yeast two-hybrid method described below.

3) Chain-Shuffling of Expression Vectors Via Cre/Loxp-MediatedSite-Specific Recombination

In a variation of the above-described methods for generating the libraryof expression vectors, the diversity of the library of expressionvectors may be increased by chain shuffling via site-specificrecombination. Accordingly, the method further comprises: causingsite-specific recombination between the members of the library of theyeast expression vectors at the 5′- and 3′-recombination sites, therecombination resulting in exchange of the first V1 or second V2nucleotide sequences between the members of the library of the yeastexpression vectors.

According to the variation, the 5′- and 3′-flanking sequences at theends of the first or second insert nucleotide sequence comprise a 5′-and 3′-recombination site, respectively, that are recognized by asite-specific recombinase.

Also according to the variation, the 5′- and 3′-site-specificrecombination sites may preferably be different site-specificrecombination sites, more preferably be sites each of which isindependently selected from the group consisting of SEQ ID Nos: 1-13,most preferably be loxP of coliphase P1, and the other be a mutant loxPsequence.

Also according to the variation, the site-specific recombinase may beconstitutively or inducibly expressed in the yeast cells. Thesite-specific recombinase may be CRE recombinase that cause thesite-specific recombination.

FIG. 4A illustrates an embodiment of this method according to thepresent invention, the library of expression vectors containing V1 andV2 are subject to mutagenesis in vivo through site-specific homologousrecombination. Each of the expression vector may contain the same yeastselection marker such as Leu 2. Through this mutagenesis, the diversityof the sequences encoding V1 and V2 can be further increased.

As illustrated in FIG. 4A, both the 5′ and the 3′ flanking sequences ofthe V2 (or V1) include a recombination site recognized by asite-specific recombinase. Preferably, the recombination site may be alox site that is recognized by the CRE recombinase of bacteriophage P1.Table 1 lists examples of lox sites that may be used in the presentinvention, including wild-type loxP sites LoxP WT [SEQ ID NO: 1] andloxP2 [SEQ ID NO: 5], and other loxP variants with mutations in the13-bp inverted repeats region and/or the 8-bp nonpalindromic core region(underlined), loxP511 [SEQ ID NO: 2], loxC2 [SEQ ID NO: 3], loxP1 [SEQID NO: 4], loxP3 [SEQ ID NO: 6], loxP4 [SEQ ID NO: 7], loxP5 [SEQ ID NO:8], loxP6 [SEQ ID NO: 9], loxP7 [SEQ ID NO: 10], loxP8 [SEQ ID NO: 11],loxP9 [SEQ ID NO: 12], and loxP10 [SEQ ID NO: 13].

More preferably, the recombination sites in the 5′ and the 3′ flankingsequences are of different lox sites, loxA and loxB as illustrated inFIG. 4A. In the presence of CRE recombinase, the expression vectorhaving the lox sites in the 5′ (loxA) and 3′ (loxB) flanking sequence ofV2 (or V1) undergoes a “chain-shuffling” with another expression vectorhaving the same lox sites in the 5′ and 3′ flanking sequence of V2′ (orV1′), respectively. As a result, the V2 chain of the expression vectoris replaced with the V2′ chain of another expression vector, therebyincreasing the complexity of the library from 10^(n) to10^(n)×10^(n)=10^(2n) theoretically.

Optionally, the recombination site may be a recombination site that isrecognized by a recombinase other than CRE. Examples of the non-CRErecombinases include, but are not limited to, site-specific recombinasesinclude: att sites recognized by the Int recombinase of bacteriophage λ(e.g. att1, att2, att3, attP, attB, attL, and attR), the FRT sitesrecognized by FLP recombinase of the 2pi plasmid of Saccharomycescerevisiae, the recombination sites recognized by the resolvase family,and the recombination site recognized by transposase of Bacillusthruingiensis.

In a preferred embodiment where V1 is V_(H) and V2 is V_(L), the scFvlibrary generated by the yeast homologous recombination is mutagenizedby a CRE/loxP mediated site-specific recombination. For example, thescFv library may comprise 10⁶ or more highly diverse and complexV-region gene repertoire derived from heavy chain and light chain originof human antibodies. One pool (e.g., the light chain gene pool V_(L)) isflanked by two non-identical Lox P sites that provide the recombinationsignals for light chain “shuffling” mediated by CRE recombinase.

The entire scFv library may be isolated from yeast and transformed intobacterial strain (e.g. KC8) through bacteria-yeast leucine nutritionalmarker complementation, and large scale DNA isolation from bacteria isperformed and the DNA pools are mixed. This pooled DNA source is thenre-introduced into yeast using conventional single plasmidtransformation protocol. The condition of this transformation maypreferably be set to enrich for multiple plasmid entry into every singleyeast cell.

The yeast cell may be pre-transformed with a plasmid that stably, ormore preferably inducibly, expresses CRE recombinase. The expression ofCRE in the yeast strain should cause the CRE-mediated homologousrecombination at the loxP sites that flank each light chain genefragment V_(L) on each expression vector. Therefore, while yeast isallowed to grow and the plasmids in the yeast cells are makingadditional copies, shuffling of the light chain gene segment occursinside of yeast cells in the presence of CRE recombinase. Therefore, thetotal number of combination of heavy chain V_(H) and light chain V_(L)within the yeast cells may be increased exponentially. Thus,theoretically, the complexity of the library can reach 10⁶×10⁶=10¹² orhigher.

One of the advantages of using the site-specific recombination in yeastis that the recombination in yeast does not require any markerselection. For example, CRE/loxP recombination will occur irrespectiveof with selection or without selection.

The multiple entry of the library of expression vectors into the yeastcells may be tested by using plasmids carrying green fluorescent protein(GFP) genes with different colors. If multiple plasmids are transformedinto a single yeast cell, certain fraction of the transformed yeast cellwill show a spectrum of combined colors. This test may also be used foroptimizing the condition for transformation of multiple expressionvectors.

Alternatively, multiple vectors from the library of expression vectorsmay be transformed into a single yeast cell by using a method of “forcedtransformation”. Under this alternative embodiment, two startinglibraries expression vectors containing V1 and V2 may be generatedseparately in two expression vectors with different selection markers.

FIG. 4B illustrates an example of this method. As illustrated in FIG.4B, two libraries of expression vectors are generated in yeast viahomologous recombination by using the procedures described above. Thetwo libraries may be otherwise the same in terms of their source RNA,amplification, and the cloning procedures. The only difference may bethe selection marker contained in the vectors.

For example, each of the expression vectors in one library contains Leu2 as a yeast selection marker, while the other contains Ade 2 as a yeastselection marker. Similar to the method illustrated in FIG. 4A, in theexpression vectors in both of two libraries, the recombination sites inthe 5′ and the 3′ flanking sequences of V2 are of different lox sites,loxA and loxB.

In the presence of CRE recombinase, the Leu 2-containing expressionvector having the lox sites in the 5′ (loxA) and 3′ (loxB) flankingsequence of V2 (or V1) undergoes a “chain-shuffling” with the Ade2-containing expression vector having the same lox sites in the 5′ and3′ flanking sequence of V2′ (or V1′), respectively. As a result, the V2chain of the expression vector is replaced with the V2′ chain of anotherexpression vector, thereby increasing the complexity of the library from10^(n) to 10^(n)×10^(n)=10^(2n) theoretically.

A combined library of both Leu 2- and Ade 2-containing expressionvectors are used to transform yeast cells, such as Y187 cells. ByLeucine and Adenine complementation in the yeast, transformants areplated in a selection medium such as SD/-Leu/-Ade medium for selectingboth types of library plasmids. Any yeast colonies formed on this doubleselection medium must have transformed by both types of library clones.

Through the selection of both markers it may be ensured that every yeastcell have both types of library clones (each may have with multiplecopies). The activation or expression of CRE enzyme in the yeast willallow the Lox P-mediated recombination.

The present invention also provides a method of producing a library ofsingle chain antibodies. In an embodiment, the method comprises:expressing in yeast cells a library of yeast expression vectors. Each ofthe yeast expression vector comprises a first nucleotide sequenceencoding an antibody heavy chain variable region, a second nucleotidesequence encoding an antibody light chain variable region, and a linkersequence encoding a linker peptide that links the antibody heavy chainvariable region and the antibody light chain variable region. Theantibody heavy chain variable region, the antibody light chain variableregion, and the linker peptide are expressed as a single fusion protein.Also, the first and second nucleotide sequences each independentlyvaries within the library of expression vectors to generate a library ofsingle-chain antibodies with a diversity of at least 10⁶.

According to the embodiment, the diversity of the library ofsingle-chain antibodies is preferably between 10⁶-10¹⁸, more preferablybetween 10⁸-10¹⁸, and most preferably between 10¹⁰-10⁸.

3. Selection of Affinity Binding Pairs between the Library of FusionProteins of the Present Invention and Target Proteins

The present invention also provides methods for screeningprotein-protein or protein-peptide binding pairs in a yeast two-hybridsystem.

The two-hybrid system is a selection scheme designed to screen forpolypeptide sequences which bind to a predetermined polypeptide sequencepresent in a fusion protein. Chien et al. (1991) Proc. Natl. Acad. Sci.(USA) 88: 9578). This approach identifies protein-protein interactionsin vivo through reconstitution of a transcriptional activator. Fieldsand Song (1989) Nature 340: 245), the yeast Gal 4 transcription protein.The method is based on the properties of the yeast Gal 4 protein, whichconsists of separable domains responsible for DNA-binding andtranscriptional activation. Polynucleotides encoding two hybridproteins, one consisting of the yeast Gal 4 DNA-binding domain (BD)fused to a polypeptide sequence of a known protein and the otherconsisting of the Gal4 activation domain (AD) fused to a polypeptidesequence of a second protein, are constructed and introduced into ayeast host cell. Intermolecular binding between the two fusion proteinsreconstitutes the Gal4 DNA-binding domain with the Gal4 activationdomain, which leads to the transcriptional activation of a reporter gene(e.g., lacZ, HIS3) which is operably linked to a Gal4 binding site.

Typically, the two-hybrid method is used to identify novel polypeptidesequences which interact with a known protein. Silver and Hunt (1993)Mol. Biol. Rep. 17: 155; Durfee et al. (1993) Genes Devel. 7; 555; Yanget al. (1992) Science 257: 680; Luban et al. (1993) Cell 73: 1067; Hardyet al. (1992) Genes Devel. 6; 801; Bartel et al. (1993) Biotechniques14: 920; and Vojtek et al. (1993) Cell 74: 205. The two-hybrid systemwas used to detect interactions between three specific single-chainvariable fragments (scFv) and a specific antigen. De Jaeger et al.(2000) FEBS Lett. 467: 316-320. The two-hybrid system was also used toscreen against cell surface proteins or receptors such as receptors ofhematopoietic super family in yeast. Ozenberger, B. A., and Young, K. H.(1995) “Functional interaction of ligands and receptors of hematopoieticsuperfamily in yeast” Mol Endocrinol. 9: 1321-1329.

Variations of the two-hybrid method have been used to identify mutationsof a known protein that affect its binding to a second known protein Liand Fields (1993) FASEB J. 7: 957; Lalo et al. (1993) Proc. Natl. Acad.Sci. (USA) 90: 5524; Jackson et al. (1993) Mol. Cell. Biol. 13; 2899;and Madura et al. (1993) J. Biol. Chem. 268: 12046.

Two-hybrid systems have also been used to identify interactingstructural domains of two known proteins or domains responsible foroligomerization of a single protein. Bardwell et al. (1993) Med.Microbiol. 8: 1177; Chakraborty et al. (1992) J. Biol. Chem. 267: 17498;Staudinger et al. (1993) J. Biol. Chem. 268: 4608; and Milne G T; WeaverD T (1993) Genes Devel. 7; 1755; Iwabuchi et al. (1993) Oncogene 8;1693; Bogerd et al. (1993) J. Virol. 67: 5030).

Variations of two-hybrid systems have been used to study the in vivoactivity of a proteolytic enzyme. Dasmahapatra et al. (1992) Proc. Natl.Acad. Sci. (USA) 89: 4159. Alternatively, an E. coli/BCCP interactivescreening system was used to identify interacting protein sequences(i.e., protein sequences which heterodimerize or form higher orderheteromultimers). Germino et al. (1993) Proc. Natl. Acad. Sci. (U.S.A.)90: 933; and Guarente L (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 1639.

Typically, selection of binding protein using a two-hybrid method reliesupon a positive association between two Gal4 fusion proteins, therebyreconstituting a functional Gal4 transcriptional activator which theninduces transcription of a reporter gene operably linked to a Gal4binding site. Transcription of the reporter gene produces a positivereadout, typically manifested either (1) as an enzyme activity (e.g.,α-galactosidase) that can be identified by a calorimetric enzyme assayor (2) as enhanced cell growth on a defined medium (e.g., HIS3 and Ade2). Thus, the method is suited for identifying a positive interaction ofpolypeptide sequences, such as antibody-antigen interactions.

False positives clones that indicate activation of the reporter geneirrespective of the specific interaction between the two hybridproteins, may arise in the two-hybrid screening. Various procedures havedeveloped to reduce and eliminate the false positive clones from thefinal positives. For example, 1) prescreening the clones that containsthe target vector and shows positive in the absence of the two-hybridpartner (Bartel, P. L., et al. (1993) “Elimination of false positivesthat arise in using the two-hybrid system” BioTechniques 14: 920-924);2) by using multiple reporters such as His3, β-galactosidase, and Ade2(James, P. et al. (1996) “Genomic libraries and a host strain designedfor highly efficient two-hybrid selection in yeast” Genetics 144:1425-1436); 3) by using multiple reporters each of which is underdifferent GAL 4-responsive promoters such as those in yeast strain Y190where each of the His 3 and β-Gal reporters is under the control of adifferent promoter Gal 1 or Gal 10, but both response to Gal 4 signaling(Durfee, T., et al (1993) “The retinoblastoma protein associates withthe protein phosphatase type 1 catalytic subunit” Genes Devel. 7:555-569); and 4) by post-screening assays such as testing isolates withtarget consisting of GAL 4-BD alone.

In addition, the false positive clones may also be eliminated by usingunrelated targets to confirm specificity. This is a standard controlprocedure in the two-hybrid system which can be performed after thelibrary isolate is confirmed by the above-described 1)-4) procedures.Typically, the library clones are confirmed by co-transforming theinitially isolated library clones back into the yeast reporter strainwith one or more control targets unrelated to the target used in theoriginal screening. Selection is conducted to eliminate those libraryclones that show positive activation of the reporter gene and thusindicate non-specfic interactions with multiple, related proteins.

The present invention provides efficient methods for screening thepolypeptide encoded by V1 and V2 in the library of expression vectorsfor their affinity binding to one or more target proteins.

According to the present invention, the method comprises: expressing alibrary of tester proteins in yeast cells, each tester protein being afusion protein comprised of a first polypeptide subunit whose sequencevaries within the library, a second polypeptide subunit whose sequencevaries within the library independently of the first polypeptide, and alinker peptide which links the first and second polypeptide subunits;expressing one or more target fusion proteins in the yeast cellsexpressing the tester proteins, each of the target fusion proteinscomprising a target peptide or protein; and selecting those yeast cellsin which a reporter gene is expressed, the expression of the reportergene being activated by binding of the tester fusion to the targetfusion protein.

According to the method, the diversity of the first or the secondpolypeptide subunit is preferably between 10³-10⁸, more preferablybetween 10⁴-10⁸ and most preferably between 10⁵-10¹⁸.

Also according to the method, the diversity of the fusion proteinsencoded by the library of expression vectors is preferably between10⁶-10¹⁸, more preferably between 10⁹-10¹⁸ and most preferably between10¹⁰-10¹⁸.

A feature of the present invention is that the first and secondpolypeptide subunits may be selected entirely independent of the targetpeptide or protein and need not be based on in any way upon one or moreproteins known to the bind to the target. As a result, the diversitiesof the first and second polypeptide subunits may be each independentlyderived from libraries of precursor sequences that are not specificallydesigned for the target peptide or protein. For example, the librariesof precursor sequences need not be derived from a small group (e.g.2-20) of genes with predetermined sequences and encoding proteins thatare known to the bind the target peptide or protein.

The diversities of the first and second polypeptide subunits also neednot be derived from one or more proteins that are known to bind to thetarget peptide or protein. For example, the one or more proteins neednot be derived from a small group (e.g. 2-20) of proteins withpredetermined sequences that are known to bind to the target peptide orprotein.

The diversities of the first and second polypeptide subunits also neednot be generated by mutagenizing one or more proteins that are known tobind to the target peptide or protein. For example, the first and secondpolypeptide subunits need not be generated by mutagenizing a small group(e.g. 2-20) of proteins with predetermined sequences and known to bindto the target peptide or protein.

In a variation of the embodiment, a single target fusion protein isexpressed and screened against the library of tester proteins. Accordingto the variation, the step of expressing the library of tester fusionproteins may include transforming a library of tester expression vectorsinto the yeast cells which contain a reporter construct comprising thereporter gene whose expression is under transcriptional control of atranscription activator comprising an activation domain and a DNAbinding domain. Each of the tester expression vectors comprises a firsttranscription sequence encoding either the activation domain AD or theDNA binding domain BD of the transcription activator, a first nucleotidesequence V1 encoding the first polypeptide subunit, a second nucleotidesequence V2 encoding the second polypeptide subunit, and a linkersequence L encoding a linker peptide that links the first nucleotidesequence and the second nucleotide sequence. Optionally, the step ofexpressing the target fusion proteins includes transforming a targetexpression vector into the yeast cells simultaneously or sequentiallywith the library of tester expression vectors. The target expressionvector comprises a second transcription sequence encoding either theactivation domain AD or the DNA binding domain BD of the transcriptionactivator which is not expressed by the library of tester expressionvectors; and a target sequence encoding the target protein or peptide.

FIG. 5 illustrates a flow diagram of a preferred embodiment of the abovedescribed method. As illustrated in FIG. 5, the sequence librarycontaining V1 and V2 fused with an AD domain upstream is carried by alibrary of expression vectors, the AD-V1-V2 vectors. The coding sequenceof the target protein (labeled as “Target”) is contained in anotherexpression vector and fused with a BD domain, forming the BD-Targetvector.

The AD-V1-V2 vector and the BD-Target vector may be co-transformed intoa yeast cell by using method known in the art. Gietz, D. et al. (1992)“Improved method for high efficiency transformation of intact yeastcells” Nucleic Acids Res. 20: 1425. The construct carrying the specificDNA binding site and the reporter gene (labeled as “Reporter”) may bestably integrated into the genome of the host cell or transientlytransformed into the host cell. Upon expression of the sequences in theexpression vectors, the library of fusion proteins comprising AD, V1 andV2, labeled as the AD-V1-V2 fusion proteins, undergo protein folding inthe host cell and adopt various conformations. Some of the AD-V1-V2fusion proteins may bind to the Target protein expressed by theBD-Target vector in the host cell, thereby bringing the AD and BDdomains to a close proximity in the promoter region (i.e., the specificDNA binding site) of the reporter construct and thus reconstituting afunctional transcription activator composed of the AD and BD domains. Asa result, the AD activates the transcription of the reporter genedownstream from the specific DNA binding site, resulting in expressionof the reporter gene, such as the lacZ reporter gene. Clones showing thephenotype of the reporter gene expression are selected, and the AD-V1-V2vectors are isolated. The coding sequences for V1 and V2 are identifiedand characterized.

Alternatively, the steps of expressing the library of tester fusionproteins and expressing the target fusion protein includes causingmating between first and second populations of haploid yeast cells ofopposite mating types. The first population of haploid yeast cellscomprises a library of tester expression vectors for the library oftester fusion proteins. Each of the tester expression vector comprises afirst transcription sequence encoding either the activation domain AD orthe DNA binding domain BD of the transcription activator, a firstnucleotide sequence V1 encoding the first polypeptide subunit, a secondnucleotide sequence V2 encoding the second polypeptide subunit, and alinker sequence L encoding a linker peptide that links the firstnucleotide sequence V1 and the second nucleotide sequence V2. The secondpopulation of haploid yeast cells comprises a target expression vector.The target expression vector comprises a second transcription sequenceencoding either the activation domain AD or the DNA binding domain BD ofthe transcription activator which is not expressed by the library oftester expression vectors; and a target sequence encoding the targetprotein or peptide. Either the first or second population of haploidyeast cells comprises a reporter construct comprising the reporter genewhose expression is under transcriptional control of the transcriptionactivator.

In this method, the haploid yeast cells of opposite mating types maypreferably be α and a type strains of yeast. The mating between thefirst and second populations of haploid yeast cells of α and a typestrains may be conducted in a rich nutritional culture medium.

FIG. 6 illustrates a flow diagram of a preferred embodiment of the abovedescribed method. As illustrated in FIG. 6, the sequence librarycontaining V1 and V2 fused with an AD domain upstream is carried by alibrary of expression vectors, the AD-V1-V2 vectors. The library of theAD-V1-V2 vectors are transformed into haploid yeast cells such as the atype strain of yeast.

The coding sequence of the target protein (labeled as “Target”) iscontained in another expression vector and fused with a BD domain,forming the BD-Target vector. The BD-Target vector is transformed intohaploid cells of opposite mating type of the haploid cells containingthe the AD-V1-V2 vectors, such as the α type strain of yeast. Theconstruct carrying the specific DNA binding site and the reporter gene(labeled as “Reporter”) may be transformed into the haploid cells ofeither the type a or type α strain of yeast.

The haploid cells of the type a and type α strains of yeast are matedunder suitable conditions such as low speed of shaking in liquidculture, physical contact in solid medium culture, and rich medium suchas YPD. Bendixen, C. et al. (1994) “A yeast mating-selection scheme fordetection of protein-protein interactions”, Nucleic Acids Res. 22:1778-1779. Finley, Jr., R. L. & Brent, R. (1994) “Interaction matingreveals lineary and ternery connections between Drosophila cell cycleregulators”, Proc. Natl. Acad. Sci. USA, 91: 12980-12984. As a result,the AD-V1-V2, the BD-Target expression vectors and the Reporterconstruct are taken into the parental diploid cells of the a and type αstrain of haploid yeast cells.

Upon expression of the sequences in the expression vectors in theparental diploid cells, the library of fusion proteins comprising AD, V1and V2, labeled as the AD-V1-V2 fusion proteins, undergo protein foldingin the host cell and adopt various conformations. Some of the AD-V1-V2fusion proteins may bind to the Target protein expressed by theBD-Target vector in the parental diploid cell, thereby bringing the ADand BD domains to a close proximity in the promoter region (i.e., thespecific DNA binding site) of the reporter construct and thusreconstituting a functional transcription activator composed of the ADand BD domains. As a result, the AD activates the transcription of thereporter gene downstream from the specific DNA binding site, resultingin expression of the reporter gene, such as the lacZ reporter gene.Clones showing the phenotype of the reporter gene expression areselected, and the AD-V1-V2 vectors are isolated. The coding sequencesfor V1 and V2 are identified and characterized.

A wide variety of reporter genes may be used in the present invention.Examples of proteins encoded by reporter genes include, but are notlimited to, easily assayed enzymes such as β-galactosidase,α-galactosidase, luciferase, β-glucuronidase, chloramphenicol acetyltransferase (CAT), secreted embryonic alkaline phosphatase (SEAP),fluorescent proteins such as green fluorescent protein (GFP), enhancedblue fluorescent protein (EBFP), enhanced yellow fluorescent protein(EYFP) and enhanced cyan fluorescent protein (ECFP); and proteins forwhich immunoassays are readily available such as hormones and cytokines.The expression of these reporter genes can also be monitored bymeasuring levels of mRNA transcribed from these genes.

When the screening of the V1 and V2 library is conducted in yeast cells,certain reporter(s) are of nutritional reporter which allows the yeastto grow on the specific selection medium plate. This is a very powerfulscreening process, as has been shown by many published papers. Examplesof the nutritional reporter include, but are not limited to, His3, Ade2,Leu2, Ura3, Trp1 and Lys2. The His3 reporter is described in Bartel, P.L. et al. (1993) “Using the two-hybrid system to detect protein-proteininteractions”, in Cellular interactions in Development: A practicalapproach, ed. Hastley, D. A., Oxford Press, pages 153-179. The Ade2reporter is described in Jarves, P. et al. (1996) “Genomic libraries anda host strain designed for highly efficient two-hybrid selection inyeast” Genetics 144: 1425-1436.

For example, a library of scFV expression vectors that contains theV_(H) and V_(L) fused with an AD domain of GAL 4 transcription activator(the AD-scFv library) may be transformed into haploid cells of the αmating type of yeast strain. A BD domain of GAL 4 transcriptionactivator is fused with the sequence encoding the target protein to beselected against the scFV library in a plasmid. This plasmid istransformed into haploid cells of the a mating type of yeast strain.

Equal volume of AD-scFv library-containing yeast stain (α-type) and theBD-target-containing yeast strain (a-type) are inoculated into selectionliquid medium and incubated separately first. These two cultures arethen mixed and allowed to grow in rich medium such as 1×YPD and 2×YPD.Under the rich nutritional culture condition, the two haploid yeaststrains will mate and form diploid cells. At the end of this matingprocess, these yeast cells are plated into selection plates. Amultiple-marker selection scheme may be used to select yeast clones thatshow positive interaction between the scFVs in the library and thetarget. For example, a scheme of SD/-Leu-Trp-His-Ade may be used. Thefirst two selections (Leu-Trp) are for markers (Leu and Trp) expressedfrom the AD-scFv library and the BD-Target vector, respectively. Throughthis dual-marker selection, diploid cells retaining both BD and ADvectors in the same yeast cells are selected. The latter two markers,His-Ade, are used to screen for those clones that express the reportergene from parental strain, presumably due to affinity binding betweenthe scFVs in the library and the target.

After the screening by co-transformation, or by mating screening asdescribed above, the putative interaction between the gene probe and thelibrary clone isolates can be further tested and confirmed in vitro orin vivo.

In vitro binding assays may be used to confirm the positive interactionbetween the tested protein expressed by the clone isolate and the targetprotein or peptide. For example, the in vitro binding assay may be a“pull-down” method, such as using GST (glutathione S-transferase)-fusedgene probe as matrix-binding protein, and with in vitro expressedlibrary clone isolate that are labeled with a radioactive ornon-radioactive group. While the probe is bound to the matrix throughGST affinity substrate (glutathione-agarose), the library clone isolatewill also bind to the matrix through its affinity with the gene probe.The in vitro binding assay may also be a Co-immuno-precipitation (Co-IP)method using two affinity tag antibodies. In this assay, both the targetgene probe and the library clone isolate are in vitro expressed fusedwith peptide tags, such as HA (haemaglutinin A) or Myc tags. The geneprobe is first immuno-precipitated with an antibody against the affinitypeptide tag (such as HA) that the target gene probe is fused with. Thenthe second antibody against a different affinity tag (such as Myc) thatis fused with the library clone isolate is used for reprobing theprecipitate.

In vivo assays may also be used to confirm the positive interactionbetween the tested protein expressed by the clone isolate and the targetprotein or peptide. For example, a mammalian two-hybrid system may serveas a reliable verification system for the yeast two-hybrid libraryscreening. In this system, the target gene probe and library clone arefused with Gal 4 DNA-binding domain or an mammalian activation domain(such as VP-16) respectively. These two fusion proteins under control ofa strong and constitutive mammalian promoter (such as CMV promoter) areintroduced into mammalian cells by transfection along with a reporterresponsive to Gal 4. The reporter can be CAT gene (chloramphenicalacetate transferase) or other commonly used reporters. After 2-3 days oftransfection, CAT assay or other standard assays will be performed tomeasure the strength of the reporter which is correlated with thestrength of interaction between the gene probe and the library cloneisolate.

The present invention also provides a kit for selecting selecting testerproteins capable of binding to a target peptide or protein.

In an embodiment, the kit comprises: a library of tester expressionvectors and a yeast cell line. Each of the tester expression vectorscomprises a first transcription sequence encoding either an activationdomain or a DNA binding domain of a transcription activator, a firstnucleotide sequence encoding a first polypeptide subunit, a secondnucleotide sequence encoding a second polypeptide subunit, and a linkersequence encoding a linker peptide that links the first nucleotidesequence and the second nucleotide sequence. The first and secondnucleotide sequences each independently varies within the library ofexpression vectors. A reporter construct may be contained in the yeastcell line. The reporter construct comprises a reporter gene whoseexpression is under a transcriptional control of a specific DNA bindingsite.

Optionally, the kit may further comprise a target expression vectorwhich comprises a second transcription sequence encoding either theactivation domain or the DNA binding domain of the transcriptionactivator which is not expressed by the library of tester expressionvectors; and a target sequence encoding the target protein or peptide.

In another embodiment, the kit comprises: a first and second populationsof haploid yeast cells of opposite mating types. The first population ofhaploid yeast cells comprises a library of tester expression vectors forthe library of tester fusion proteins. Each of the tester expressionvector comprises a first transcription sequence encoding either anactivation domain or a DNA binding domain of a transcription activator,a first nucleotide sequence encoding a first polypeptide subunit, asecond nucleotide sequence encoding a second polypeptide subunit, and alinker sequence encoding a linker peptide that links the firstnucleotide sequence and the second nucleotide sequence. The secondpopulation of haploid yeast cells comprises a target expression vector.The target expression vector encodes either the activation domain or theDNA binding domain of the transcription activator which is not expressedby the library of tester expression vectors; and a target sequenceencoding the target protein or peptide. Either the first or secondpopulation of haploid yeast cells comprises a reporter constructcomprising a reporter gene whose expression is under transcriptionalcontrol of the transcription activator.

Optionally, the second population of haploid yeast cells comprises aplurality of target expression vectors. Each of the target expressionvectors encodes either the activation domain or the DNA binding domainof the transcription activator which is not expressed by the library oftester expression vectors; and a target sequence encoding the targetprotein or peptide. Either the first or second population of haploidyeast cells comprises a reporter construct comprising a reporter genewhose expression is under transcriptional control of the transcriptionactivator.

4. Selection of Affinity Binding Pairs between the Library of FusionProteins of the Present Invention and Target Nucleic Acids

As described above, the libraries of V1 and V2 sequences of the presentinvention can be used for selecting protein-protein or protein-peptidebinding pairs against single or arrayed multiple protein/peptide targetsin a two-hybrid screening system. As described in the following, theselibraries can also be used for selecting protein-DNA or protein-RNAbinding pairs in an one-hybrid system or three-hybrid system,respectively.

The general scheme for screening protein-DNA binding pair using anone-hybrid system is described in Li and Herskowitz (1993) Science262:1870-1874. Typically, this method is used to identify genes encodingproteins that recognize a specific DNA sequence. A library of randomprotein segments tagged with a transcriptional activation domain (AD) isscreened for proteins that can activate a reporter gene containing thespecific DNA sequence in its promoter region. By using this strategy, anessential protein that interacts in vivo with the yeast origin of DNAreplication was identified. In a three-hybrid system, the target nucleicacid is RNA or RNA-associated proteins. SanGupta, et al. (1996) Proc.Natl. Acad. Sci. USA 93:8496-8501.

The present invention provides a method is provided for screeningprotein-DNA binding pairs in a yeast one-hybrid system.

In an embodiment, the method comprises: expressing a library of testerfusion proteins in yeast cells which contain a reporter constructcomprising a reporter gene whose expression is under a transcriptionalcontrol of a target DNA sequence; and selecting the yeast cells in whichthe reporter gene is expressed, the expression of the reporter genebeing activated by binding of the tester fusion protein to the targetDNA sequence. Each of the tester fusion proteins comprises an activationdomain AD of a transcription activator, a first polypeptide subunitwhose sequence varies within the library, a second polypeptide subunit,whose sequence varies within the library independently of the firstpolypeptide subunit, and a linker peptide that links the firstpolypeptide subunit to the second polypeptide subunit.

In a variation of the embodiment, the step of expressing the library oftester fusion proteins includes transforming into the yeast cells alibrary of tester expression vectors for the library of tester fusionproteins. Each of the tester expression vectors comprises atranscription sequence encoding the activation domain AD of thetranscription activator, a first nucleotide sequence V1 encoding thefirst polypeptide subunit, a second nucleotide sequence V2 encoding thesecond polypeptide subunit, and a linker sequence L encoding a linkerpeptide that links the first nucleotide sequence V1 and the secondnucleotide sequence V2.

In another variation of the embodiment, the step of expressing a libraryof tester fusion proteins in yeast cells includes causing mating betweena first and second populations of haploid yeast cells of opposite matingtypes. The first population of haploid yeast cells comprises a libraryof tester expression vectors for the library of tester fusion proteins,each tester expression vector comprising a transcription sequenceencoding the activation domain AD of the transcription activator, afirst nucleotide sequence V1 encoding the first polypeptide subunit, asecond nucleotide sequence V2 encoding the second polypeptide subunit,and a linker sequence L encoding a linker peptide that links the firstnucleotide sequence V1 and the second nucleotide sequence V2. The secondpopulation of haploid yeast cells comprises the reporter construct.

According to the variation, the haploid yeast cells of opposite matingtypes may preferably be α and a type strains of yeast. The matingbetween the first and second populations of haploid yeast cells of α anda type strains may preferably conducted in a rich nutritional culturemedium.

According to any of the above-described methods for selectingprotein-DNA binding pairs, the target DNA sequence in the reporterconstruct may preferably be positioned in 2-6 tandem repeats 5′ relativeto the reporter gene.

The target DNA sequence in the reporter construct may be preferablybetween about 15-75 bp in length and more preferably between about 25-55bp in length.

FIG. 7 illustrates a flow diagram of a preferred embodiment of theabove-described method. As illustrated in FIG. 7, the tester sequencelibrary containing V1 and V2 fused with an AD domain upstream is carriedby a library of expression vectors, the AD-V1-V2 vector. The target DNAsequence (labeled “Target DNA”) is positioned in the promoter region ofa reporter gene (labeled “Reporter”).

The AD-V1-V2 vector is transformed into a yeast cell by using methodsknown in the art. Gietz, D. et al. (1992) “Improved method for highefficiency transformation of intact yeast cells” Nucleic Acids Res. 20:1425. The construct carrying the target DNA sequence and the reportergene may be stably integrated into the genome of the host cell ortransiently transformed into the host cell.

As illustrated in FIG. 7, upon expression of the tester sequences in theexpression vectors, the library of tester proteins comprising AD, V1 andV2, labeled as the AD-V1-V2 fusion proteins, undergo protein folding inthe host cell and adopt various conformations. Some of the AD-V1-V2fusion proteins may bind to the target DNA sequence in the promoterregion of the reporter gene, thereby bringing the AD domain to a closeproximity in the promoter region. As a result, the AD activates thetranscription of the reporter gene downstream from the target DNAsequence, resulting in expression of the reporter gene, such as the lacZreporter gene. Clones showing the phenotype of the reporter geneexpression are selected, and the AD-V1-V2 vectors are isolated. Thecoding sequences for V1 and V2 are identified and characterized.

Alternatively, the AD-V1-V2 vector and the reporter construct may beintroduced a diploid yeast cell by mating between two haploid yeaststrains. For example, the AD-V1-V2 vector may be transformed into ahaploid yeast strain such as the α strain; and the reporter constructmay be transformed into another haploid yeast strain such as the s the astrain. Upon mating between these two haploid strains, diploid cells areformed to merge the genetic materials carried by the two haploid cells.As a result, the AD-V1-V2 vector and the reporter construct areintroduced into a diploid cell which is then screened for positiveinteractions between the tester protein and the target DNA in the cell.

The target DNA sequence may be a regulatory element, or a putativechromosome remodeling protein complex opening site, preferably in ashort stretch of DNA sequence (20-80 bp). The target DNA sequence may becloned into a yeast one-hybrid system reporter vector, e.g., PHIS(Clontech, Palo Alto, Calif.; Luo et al. (1996) “Cloning and analysis ofDNA-binding proteins by yeast one-hybrid and one-two-hybrid system”Biotechniques 20: 564-568). To increase the sensitivity, the targetsequence may be cloned as in a few tandem repeats (e.g., 4-5 copies)into the reporter vector. The recombinant reporter vector may beintegrated into the yeast reporter strain by a transformation withlinearized vector and selection for rescuing the integration marker. Theintegration should be at a single chromosome location and usually athigh efficiency.

The tester sequence library containing V1 and V2 may encode a library ofscFv that can be used to screen against a target DNA antigen. The scFvexpression library may be introduced into yeast by transformation or bymating with the yeast strain of the opposite mating type and harboringthe reporter construct. The transformation and mating procedures aredescribed in detail in Example 3. Pre-screening of self-activatingclones may be necessary for eliminating the false positive clones. Theprocedures are similar to the two-hybrid library pre-screening describedin Section 3.

The library clones isolated from such a one-hybrid system screening mayindicate that scFv antibody(s) expressed from these clones are capableof binding to the DNA target. Such antibody may be have significantapplications in DNA vaccine and diagnostics of diseases.

The one-hybrid system of the present invention may also be modified toscreen for novel co-factors that bind to a known DNA-binding factor. Thelibrary of polypeptides comprising V1 and V2 subunits fused with an ADdomain may be screened for affinity binding toward a specific factorthat binds to a DNA sequence in the promoter region of a reporter gene.

In one embodiment, the method comprises: expressing a library of testerfusion proteins in yeast cells which contain a reporter constructcomprising a reporter gene whose expression is under a transcriptionalcontrol of a specific DNA binding site; expressing a target protein inthe yeast cells expressing the tester fusion proteins, where the targetprotein binds to the specific DNA binding site; and selecting the yeastcells in which the reporter gene is expressed, the expression of thereporter gene being activated by binding of the tester fusion protein tothe target protein. Each of the tester fusion proteins comprises anactivation domain AD of a transcription activator, a first polypeptidesubunit, a second polypeptide subunit, and a linker peptide that linksthe first polypeptide subunit to the second polypeptide subunit, whereinthe sequences of the first and second polypeptide subunits eachindependently varies within the library of the tester fusion protein.

In a variation of the embodiment, the step of expressing the library oftester fusion proteins includes transforming into the yeast cells alibrary of tester expression vectors for the library of tester fusionproteins. Each of the tester expression vectors comprises atranscription sequence encoding the activation domain AD of thetranscription activator, a first nucleotide sequence V1 encoding thefirst polypeptide subunit, a second nucleotide sequence V2 encoding thesecond polypeptide subunit, and a linker sequence L encoding a linkerpeptide that links the first nucleotide sequence V1 and the secondnucleotide sequence V2.

In another variation of the embodiment, the steps of expressing thelibrary of tester fusion proteins and expressing the target fusionprotein includes causing mating between a first and second populationsof haploid yeast cells of opposite mating types. The first population ofhaploid yeast cells comprises a library of tester expression vectors forthe library of tester fusion proteins. Each of the tester expressionvectors comprises a transcription sequence encoding the activationdomain AD of the transcription activator, a first nucleotide sequence V1encoding the first polypeptide subunit, a second nucleotide sequence V2encoding the second polypeptide subunit, and a linker sequence Lencoding a linker peptide that links the first nucleotide sequence V1and the second nucleotide sequence V2. The second population of haploidyeast cells comprises a target expression vector comprising a targetsequence encoding the target protein. Either the first or secondpopulation of haploid yeast cells comprises the reporter construct.

FIG. 8 illustrates a flow diagram of a preferred embodiment of theabove-described method. As illustrated in FIG. 8, the tester sequencelibrary containing V1 and V2 fused with an AD domain upstream is carriedby a library of expression vectors, the AD-V1-V2 vector. The AD-V1-V2vectors are introduced into host cells, for example, by transformation.The target protein (labeled “Target”) that is known to bind to aspecific DNA sequence may be expressed by an expression vector in thehost cells or otherwise present in the cells. The specific DNA sequence(labeled “*DNA”) is positioned in the promoter region of a reporter gene(labeled “Reporter”). The construct carrying the specific DNA sequenceand the reporter gene may be stably integrated into the genome of thehost cell or transiently transformed into the host cell.

As illustrated in FIG. 8, upon expression of the tester sequences in theexpression vectors, the library of tester proteins comprising AD, V1 andV2, labeled as the AD-V1-V2 fusion proteins, undergo protein folding inthe host cell and adopt various conformations. Some of the AD-V1-V2fusion proteins may bind to the target protein that binds to thespecific DNA sequence in the promoter region of the reporter gene,thereby bringing the AD domain to a close proximity in the promoterregion. As a result, the AD activates the transcription of the reportergene downstream from the target DNA sequence, resulting in expression ofthe reporter gene, such as the lacZ reporter gene. Clones showing thephenotype of the reporter gene expression are selected, and the AD-V1-V2vectors are isolated. The coding sequences for V1 and V2 are identifiedand characterized.

The specific target protein may be any protein that has beencharacterized to be a DNA-binding fact by using various assays such asin vitro gel shifting assays, or through conventional one-hybridscreening. The target protein (without being fused to an AD domain) maybe expressed in the yeast one-hybrid reporter strain. The level oftarget protein expression is then adjusted to such an extent that nomeasurable activation is observed. The yeast strain may also contain thereporter construct that is integrated into the yeast genome.

The tester sequence library containing V1 and V2 may encode a library ofscFv that can be used to screen against a target protein that aDNA-binding factor. The library clones isolated from such a modifiedone-hybrid system screening may indicate that scFv antibody(s) expressedfrom these clones are capable of binding to the protein target. Suchantibody may be have significant applications in therapeutics anddiagnostics of diseases.

5. High Throughput Selection of Affinity Binding Pairs between theLibrary of Fusion Proteins of the Present Invention and a Library ofTarget Proteins

The present invention also provides a method for high throughputscreening of the above-described libraries of fusion proteins encoded byV1 and V2. The library of expression vectors, for example, the AD-scFvyeast expression vector library, may be screen for the binding of thescFvs to multiple target proteins expressed by a yeast clone library(BD-Target library), each clone carrying a BD-Target vector for eachtarget protein to be selected against. The BD-Target clone library maybe arrayed in multiple-well plates, such as 96- and 384-well plates, andthen screened against the scFv library in an automated and highthroughput manner.

For example, a collection of EST clones (or a total library of EST) fromhuman, mouse or other organisms may be screened against the scFv librarygenerated by using the methods of the present invention. Such acollection of EST clones may be ordered from a public resource in alibrary format with individually clones arrayed in 96-well or 384-wellplates. Lennon, G. et al. (1996) “The I.M.A.G.E. Consortium: anintegrated molecular analysis of genomes and their expression” Genomics33: 151-152. The EST inserts from the original collection (usually inbacterial cloning and sequencing vectors) may be PCR amplified withextended homologous sequences at both ends following similar proceduresused in the generation of the scFv library. Through the same homologousrecombination procedure as used in the generation of the scFv library,the EST inserts are inserted into an expression vector containing a BDdomain of a transcription activator in yeast cells.

Optionally, a collection of certain domain structures, such as zincfinger and helix-loop-helix protein domains, may be inserted into theAD-containing expression vector in yeast cell via homologousrecombination. The yeast clones containing the vector with BD fused toeach domain structure may be arrayed in multiple-well plates andscreened against the scFv library for affinity binding between the scFvand each domain structure. The domain structure may be 18-20 amino acidsat length and its sequence may not be totally random. Such a collectionof domain structures may be generated by using syntheticoligonucleotides with characteristic conserved and random/degenerateresidues to cover most of the rational domain structures.

Also optionally, the coding sequences of a random peptide library may beinserted into the BD-containing expression vector in yeast cell viahomologous recombination. The yeast clones containing the vector with ADfused to each random peptide may be arrayed in multiple-well plates andscreened against the scFv library for affinity binding between the scFvand each random peptide target. The random peptide may be 16-20 aminoacid at length. Such a library of random peptide can generated by randomoligonucleotide synthesis or by partially random oligonucleotidesynthesis biased toward a sequence encoding a specific target.

Alternatively, a library of short peptides may also be may be insertedinto the BD-containing expression vector in yeast cell via homologousrecombination. Accordingly, the scFv library may be fused with the ADdomain in the expression vector and screened against this library ofshort peptide. Through this selection, peptide ligands may be selectedfor each scFv. Strutural and functional analysis of the selectedpeptides should aid in the rational design of antigens and structuralimprovement of specific target antigens.

FIG. 9 depicts a general scheme of high throughput screening of the scFvlibrary against a library of target proteins in yeast via mating of twostrains of yeast haploid cells.

As illustrated in FIG. 9, the each member of the library of targetproteins or peptides is fused with the DB domain of an expression vectorcontained in yeast a-type of host strain.

The yeast clones of the library of target proteins may be arrayed as aclone library. This may be achieved by depositing each clone containingthe BD-Target fusion into a well of a 96- or 384-well plate. Optionally,prior to using this library of BD-Target clones, the BD-Target librarymay be preselected to filter out any self-activating clones. Thisselection may be accomplished by allowing the yeast clones that containthe BD-Target fusion to grow in a selection medium used for two-hybridselection at a later stage, such as the medium SD/-Trp-His. The clonesare checked for self-activation of the reporter gene in the absence ofthe AD domain.

Alternatively, the BD-Target library may be preselected in a selectionmedium with β- or α-galactosidase substrate. Any positive clones willproduce a colored reaction catalyzed the galactosidase expressed from aLacZ reporter gene and can be easily detected by naked eyes or by aninstrument. Such clones are self-activating clones that express thereporter gene in the absence of the AD domain. The clones may beexcluded from the library of BD-Target clones.

Still referring to FIG. 9, the BD-target clones of a-strain of yeast maybe inoculated into a plate which is pre-seeded with an arrayed libraryof scFv library of α-strain of yeast haploid cells. The two haploidyeast strains mate in the rich medium and form diploid. The parentalclones are screened for expression of the reporter gene which indicatespositive interactions between a scFV and a target protein expressed bythe clones in the same well. The scoring of the positive clones may beconveniently carried out by machine-aided automatic screening using β-or α-galactosidase substrate. Aho, S. et al. (1997) “A novel reportergene MEL1 for the yeast two-hybrid system” Anal. Biochem. 253: 270-272.

Compared to the screening of a single target protein against a libraryof scFv proteins, the method illustrated in FIG. 9 is based on a clonalmating, i.e., individual target protein against individual scFv protein.The advantage of such clonal mating is that the efficiency of mating andselection may be enhanced through clonal mating when large numbers oftarget proteins and scFv antibodies are involved.

The methods described can be used for large scale screening of librariesof biomolecules, such as fully human antibody repertoires, against awide variety target molecules or ligands. The screening process may beautomated for high throughput screening of the biomolecules. Forexample, such screening process allows for efficient isolation andcollection of scFv antibodies against any EST (human, mouse, or anyother organisms), or any known structural/functional protein domains(Zinc finger, helix-loop-helix, etc.), or totally random peptides withvarious lengths.

In contrast, by using conventional methods for screening antibody invivo, such as the hybridoma and “XENOMOUSE” technologies, such alarge-scale and comprehensive antibody collection may have beenimpractical due to technical limitations associated with using animal asthe host for the libraries of antibodies and target molecules.

By using the method of the present invention, the antibody repertoirescan be screened for affinity interaction between an antibody in thelibrary and a target antigen individually in vivo by clonal matingwithout losing track of individual clones. The screening should be moreefficient than the procedure performed on mice, owing the to fastproliferation rate and ease of handling of yeast cells.

The method of the present invention should provide vary useful tools forprofiling functions of genes, in particular, functional proteomics,efficiently and economically. With the completion of human genomesequencing, the demands are tremendous for efficient large-scalescreening for functional proteins aimed at large numbers of targetmolecules. The high affinity and functional scFv antibodies, as well asother multimeric proteins, that are selected by using the methods of thepresent invention should find a wide variety applications in prevention,diagnosis, therapeutic treatment of diseases and in other biomedical orindustrial uses.

6. Mutagenesis of the Fusion Protein Leads Positively Selected AgainstTarget Protein(s)

As described above, protein leads, such as scFv antibody leads, can beidentified through the selection of the primary library carrying V1 andV2 against one or more target proteins. The coding sequences of theseprotein leads may be mutagenized in vitro or in vivo to generated asecondary library more diverse than these leads. The mutagenized leadscan be selected against the target protein(s) again in vivo followingsimilar procedures described for the selection of the primary librarycarrying V1 and V2. Such mutagenesis and selection of primary antibodyleads effectively mimics the affinity maturation process naturallyoccurring in a mammal that produces antibody with progressive increasein the affinity to the immunizing antigen.

The coding sequences of the fusion protein leads may be mutagenized byusing a wide variety of methods. Examples of methods of mutagenesisinclude, but are not limited to site-directed mutagenesis, error-pronePCR mutagenesis, cassette mutagenesis, random PCR mutagenesis, DNAshuffling, and chain shuffling.

Site-directed mutagenesis or point mutagenesis may be used to graduallychange the V1 and V2 sequences in specific regions. This is generallyaccomplished by using oligonucleotide-directed mutagenesis. For example,a short sequence of a scFv antibody lead may be replaced with asynthetically mutagenized oligonucleotide. The method may not beefficient for mutagenizing large numbers of V1 and V2 sequences, but maybe used for fine toning of a particular lead to achieve higher affinitytoward a specific target protein.

Cassette mutagenesis may also be used to mutagenize the V1 and V2sequences in specific regions. In a typical cassette mutagenesis, asequence block, or a region, of a single template is replaced by acompletely or partially randomized sequence. However, the maximuminformation content that can be obtained may be statistically limited bythe number of random sequences of the oligonucleotides. Similar to pointmutagenesis, this method may also be used for fine toning of aparticular lead to achieve higher affinity toward a specific targetprotein.

Error-prone PCR, or “poison” PCR, may be used to the V1 and V2 sequencesby following protocols described in Caldwell and Joyce (1992) PCRMethods and Applications 2: 28-33. Leung, D. W. et al. (1989) Technique1: 11-15. Shafikhani, S. et al. (1997) Biotechniques 23: 304-306.Stemmer, W. P. et al. (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751.

FIG. 10 illustrates an example of the method of the present inventionfor affinity maturation of antibody leads selected from the primary scFvlibrary. As illustrated in FIG. 10, the coding sequences of the scFvleads selected from clones containing the primary scFv library aremutagenized by using a poison PCR method. Since the coding sequences ofthe scFV library are contained in the expression vectors isolated fromthe selected clones, one or more pairs of PCR primers may be used tospecifically amplify the V_(H) and V_(L) region out of the vector. ThePCR fragments containing the V_(H) and V_(L) sequences are mutagenizedby the poison PCR under conditions that favors incorporation ofmutations into the product.

Such conditions for poison PCR may include a) high concentrations ofMn²+ (e.g. 0.4-0.6 mM) that efficiently induces malfunction of Taq DNApolymerase; and b) disproportionally high concentration of onenucleotide substrate (e.g., dGTP) in the PCR reaction that causesincorrect incorporation of this high concentration substrate into thetemplate and produce mutations. Additionally, other factors such as, thenumber of PCR cycles, the species of DNA polymerase used, and the lengthof the template, may affect the rate of mis-incorporation of “wrong”nucleotides into the PCR product. Commercially available kits may beutilized for the mutagenesis of the selected scFv library, such as the“Diversity PCR random mutagenesis kit” (catalog No. K1830-1, Clontech,Palo Alto, Calif.).

The PCR primer pairs used in mutagenesis PCR may preferably includeregions matched with the homologous recombination sites in theexpression vectors. This design allows re-introduction of the PCRproducts after mutagenesis back into the yeast host strain again viahomologous recombination. This also allows the modified V_(H) and V_(L)region to be fused with the AD domain directly in the expression vectorin the yeast.

Still referring to FIG. 10, the mutagenized scFv fragments are insertedinto the expression vector containing an AD domain via homologousrecombination in haploid cells of α type yeast strain. Similarly to theselection of scFv clones from the primary antibody library, the AD-scFvcontaining haploid cells are mated with haploid cells of opposite matingtype (e.g. a type) that contains the BD-Target vector and the reportergene construct. The parental diploid cells are selected based onexpression of the reporter gene and other selection criteria asdescribed in detail in Section 3.

Other PCR-based mutagenesis method can also be used, alone or inconjunction with the poison PCR described above. For example, the PCRamplified V_(H) and V_(L) segments may be digested with DNase to createnicks in the double DNA strand. These nicks can be expanded into gaps byother exonucleases such as Bal 31. The gaps may be then be filled byrandom sequences by using DNA Klenow polymerase at low concentration ofregular substrates dGTP, dATP, dTTP, and dCTP with one substrate (e.g.,dGTP) at a disproportionately high concentration. This fill-in reactionshould produce high frequency mutations in the filled gap regions. Thesemethod of DNase I digestion may be used in conjunction with poison PCRto create highest frequency of mutations in the desired V_(H) and V_(L)segments.

The PCR amplified V_(H) and V_(L) segments or the scFv segmentsamplified from the primary antibody leads may be mutagenized in vitro byusing DNA shuffling techniques described by Stemmer (1994) Nature 370:389-391; and Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751.The V_(H), V_(L) or scFV segments from the primary antibody leads aredigested with DNase I into random fragments which are then reassembledto their original size by homologous recombination in vitro by using PCRmethods. As a result, the diversity of the library of primary antibodyleads are increased as the numbers of cycles of molecular evolutionincrease in vitro.

The V_(H), V_(L) or scFv segments amplified from the primary antibodyleads may also be mutagenized in vivo by exploiting the inherent abilityof mution in pre-B cells. The Ig gene in pre-B cells is specificallysusceptible to a high-rate of mutation in the development of pre-Bcells. The Ig promoter and enhancer facilitate such high rate mutationsin a pre-B cell environment while the pre-B cells proliferate.Accordingly, V_(H) and V_(L) gene segments may be cloned into amammalian expression vector that contains human Ig enhancer andpromoter. This construct may be introduced into a pre-B cell line, suchas 38B9, which allows the mutation of the V_(H) and V_(L) gene segmentsnaturally in the pre-B cells. Liu, X., and Van Ness, B. (1999) Mol.Immunol. 36: 461-469. The mutagenized V_(H) and V_(L) segments can beamplified from the cultured pre-B cell line and re-introduced back intothe AD-containing yeast strain via, for example, homologousrecombination.

The secondary antibody library produced by mutagenesis in vitro (e.g.PCR) or in vivo, i.e., by passing through a mammalian pre-B cell linemay be cloned into an expression vector and screened against the sametarget protein as in the first round of screening using the primaryantibody library. For example, the expression vectors containing thesecondary antibody library may be transformed into haploid cells of αtype yeast strain. These α cells are mated with haploid cells a typeyeast strain containing the BD-target expression vector and the reportergene construct. The positive interaction of scFvs from the secondaryantibody library is screened by following similar procedures asdescribed for the selection of the primary antibody leads in yeast.

Alternatively, since the secondary antibody library may be relativelylow in complexity (e.g., 10⁴-10⁵ independent clones) as compared to theprimary libraries (e.g., 10⁷-10¹⁴), the screening of the secondaryantibody library may be performed without mating between two yeaststrains. Instead, the linearized expression vectors containing the ADdomain and the mutagenized V_(H) and V_(L) segments may be directlyco-transformed into yeast cells containing the BD-target expressionvector and the reporter gene construct. Via homologous recombination inyeast, the secondary antibody library are expressed by the recombinedAD-scFv vector and screened against the target protein expressed by theBD-target vector by following similar procedures as described for theselection of the primary antibody leads in yeast.

7. Functional Expression and Purification of Selected Antibody

The library of fusion protens encoded by V1 and V2 that are generatedand selected in the screening against the target protein(s) may beexpressed in hosts after the V1 and V2 sequences are operably linked toan expression control DNA sequence, including naturally-associated orheterologous promoters, in an expression vector. By operably linking theV1 and V2 sequences to an expression control sequence, the V1 and V2coding sequences are positioned to ensure the transcription andtranslation of these inserted sequences. The expression vector may bereplicable in the host organism as episomes or as an integral part ofthe host chromosomal DNA. The expression vector may also containselection markers such as antibiotic resistance genes (e.g. neomycin andtetracycline resistance genes) to permit detection of those cellstransformed with the expression vector.

Preferably, the expression vector may be a eukaryotic vector capable oftransforming or transfecting eukaryotic host cells. Once the expressionvector has been incorporated into the appropriate host cells, the hostcells are maintained under conditions suitable for high level expressionof the single-chains polypeptide encoded by V1 and V2, such as scFvs.The polypeptides expressed are collected and purified depending on theexpression system used.

The scFv, Fab, or fully assembled antibodies selected by using themethods of the present invention may be expressed in various scales inany host system. FIG. 12 illustrates examples of host systems: bacteria(e.g. E. coli), yeast (e.g. S. cerevisiae), and mammalian cells (COS).The bacteria expression vector may preferably contain the bacterialphage T7 promoter and express a single chain variable fragment (scFv).The yeast expression vector may contain a constitutive promoter (e.g.ADGI promoter) or an inducible promoter such as (e.g. GCN4 and Gal 1promoters). All three types of antibody, scFv, Fab, and full antibody,may be expressed in a yeast expression system.

The expression vector may be a mammalian express vector that can be usedto express the single-chains polypeptide encoded by V1 and V2 inmammalian cell culture transiently or stably. Examples of mammalian celllines that may be suitable of secreting immunoglobulins include, but arenot limited to, various COS cell lines, HeLa cells, myeloma cell lines,CHO cell lines, transformed B-cells and hybridomas.

Typically, a mammalian expression vector includes certain expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, as well as necessary processing signals, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Examples of promoters include, butare not limited to, insulin promoter, human cytomegalovirus (CMV)promoter and its early promoter, simian virus SV40 promoter, Roussarcoma virus LTR promoter/enhancer, the chicken cytoplasmic β-actinpromoter, promoters derived from immunoglobulin genes, bovine papillomavirus and adenovirus.

One or more enhancer sequence may be included in the expression vectorto increase the transcription efficiency. Enhancers are cis-actingsequences of between 10 to 300 bp that increase transcription by apromoter. Enhancers can effectively increase transcription whenpositioned either 5′ or 3′ to the transcription unit. They may also beeffective if located within an intron or within the coding sequenceitself. Examples of enhancers include, but are not limited to, SV40enhancers, cytomegalovirus enhancers, polyoma enhancers, the mouseimmunoglobulin heavy chain enhancer and adenovirus enhancers. Themammalian expression vector may also typically include a selectablemarker gene. Examples of suitable markers include, but are not limitedto, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene(TK), or prokaryotic genes conferring antibiotic resistance. The DHFRand TK genes prefer the use of mutant cell lines that lack the abilityto grow without the addition of thymidine to the growth medium.Transformed cells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

The expression vectors containing the V1 and V2 sequences can then betransferred into the host cell by methods known in the art, depending onthe type of host cells. Examples of transfection techniques include, butare not limited to, calcium phosphate transfection, calcium chloridetransfection, lipofection, electroporation, and microinjection.

The V1 and V2 sequences may also be inserted into a viral vector such asadenoviral vector that can replicate in its host cell and produce thepolypeptide encoded by V1 and V2 in large amounts.

In particular, as illustrated in FIG. 12, the scFv, Fab, or fullyassembled antibody may be expressed in mammalian cells by using a methoddescribed by Persic et al. (1997) Gene, 187: 9-18. The mammalianexpression vector that is described by Persic and contains EF-α promoterand SV40 replication origin is preferably utilized. The SV40 originallows a high level of transient expression in cells containing large Tantigen such as COS cell line. The expression vector may also includesecretion signal and different antibiotic markers (e.g. neo and hygro)for integration selection.

Once expressed, polypeptides encoded by V1 and V2 may be isolated andpurified by using standard procedures of the art, including ammoniumsulfate precipitation, fraction column chromatography, and gelelectrophoresis. Once purified, partially or to homogeneity as desired,the polypeptides may then be used therapeutically or in developing,performing assay procedures, immunofluorescent stainings, and in otherbiomedical and industrial applications. In particular, the antibodiesgenerated by the method of the present invention may be used fordiagnosis and therapy for the treatment of various diseases such ascancer, autoimmune diseases, or viral infections.

In a preferred embodiment, the scFv human antibody with V_(H) and V_(L)segments that are generated and screened by using the methods of thepresent invention may be expressed directly in yeast. According to thisembodiment, the V_(H) and V_(L) regions from the selected expressionvectors may be PCR amplified with primers that simultaneously addappropriate homologous recombination sequences to the PCR products.These PCR segments of V_(H) and V_(L) may then be introduced into ayeast strain together with a linearized expression vector containingdesirable promoters, expression tags and other transcriptional ortranslational signals.

For example, the PCR segments of V_(H) and V_(L) regions may behomologously recombined with a yeast expression vector that alreadycontains a desirable promoter in the upstream and stop codons andtranscription termination signal in the downstream. The promoter may bea constitutive expression promoter such as ADH1, or an inducibleexpression promoter, such as Gal 1, or GCN4 (A. Mimran, I. Marbach, andD. Engelberg, (2000) Biotechniques 28: 552-560). The latter induciblepromoter may be preferred because the induction can be easily achievedby adding 3-AT into the medium.

The yeast expression vector to be used for expression of the scFvantibody may be of any standard strain with nutritional selectionmarkers, such as His 3, Ade 2, Leu 2, Ura 3, Trp 1 and Lys 2. The markerused for the expression of the selected scFv may preferably be differentfrom the AD vector used in the selection of scFv in the two-hybridsystem. This may help to avoid potential carryover problem associatedwith multiple yeast expression vectors.

For expressing the scFv antibody in a secreted form in yeast, theexpression vector may include a secretion signal in the 5′ end of theV_(H) and V_(L) segments, such as an alpha factor signal and a 5-phosecretion signal. Certain commercially available vectors that contain adesirable secretion signal may also be used (e.g., pYEX-S1, catalog #6200-1, Clontech, Palo Alto, Calif.).

The scFv antibody fragments generated may be analyzed and characterizedfor their affinity and specificity by using methods known in the art,such as ELISA, western, and immune staining. Those scFv antibodyfragments with reasonably good affinity (with dissociation constantpreferably above 10⁻⁶ M) and specificity can be used as building blocksin Fab expression vectors, or can be further assembled with the constantregion for full length antibody expression. These fully assembled humanantibodies may also be expressed in yeast in a secreted form.

FIG. 11 illustrates the secondary structures of the scFv, Fab and afully assembled antibody. The V_(H) sequence encoding the selected scFvprotein may be linked with the constant regions of a full antibody, CH1,CH2 and CH3. Similarly, the VL sequence may be linked with the constantregion CL. The assembly of two units of VH-CH1—CH2-CH3 and VL-CL leadsto formation of a fully functional antibody. The present inventionprovides a method for producing fully functional antibody in yeast.Fully functional antibody retaining the rest of the constant regions mayhave a higher affinity (or avidity) than a scFv or a Fab. The fullantibody should also have a higher stability, thus allowing moreefficient purification of antibody protein in large scale.

The method is provided by exploiting the ability of yeast cells touptake and maintain multiple copies of plasmids of the same replicationorigin. According to the method, different vectors may be used toexpress the heavy chain and light chain separately, and yet allows forthe assembly of a fully functional antibody in yeast. This approach hasbeen successfully used in a two-hybrid system design where the BD and ADvectors are identical in backbone structure except the selection markersare distinct. This approach has been used in a two-hybrid system designfor expressing both BD and AD fusion proteins in the yeast. The BD andAD vectors are identical in their backbone structures except theselection markers are distinct. Both vectors can be maintained in yeastin high copy numbers. Chien, C. T., et al. (1991) “The two-hybridsystem: a method to identify and clone genes for proteins that interactwith a protein of interest” Proc. Natl. Acad. Sci. USA 88: 9578-9582.

In the present invention, the heavy chain gene and light chain genes areplaced in two different vectors. Under a suitable condition, theVH-CH1-CH2-CH3 and VL-CL sequences are expressed and assembled in yeast,resulting in a fully functional antibody protein with two heavy chainsand two light chains. This fully functional antibody may be secretedinto the medium and purified directly from the supernatant.

The scFv with a constant region, Fab, or fully assembled antibody can bepurified using methods known in the art. Conventional techniquesinclude, but are not limited to, precipitation with ammonium sulfateand/or caprylic acid, ion exchange chromatography (e.g. DEAE), and gelfiltration chromatography. Delves (1997) “Antibody Production: EssentialTechniques”, New York, John Wiley & Sons, pages 90-113. Affinity-basedapproaches using affinity matrix based on Protein A, Protein G orProtein L may be more efficiency and results in antibody with highpurity. Protein A and protein G are bacterial cell wall proteins thatbind specifically and tightly to a domain of the Fc portion of certainimmunoglobulins with differential binding affinity to differentsubclasses of IgG. For example, Protein G has higher affinities formouse IgG1 and human IgG3 than does Protein A. The affinity of Protein Aof IgG1 can be enhanced by a number of different methods, including theuse of binding buffers with increased pH or salt concentration. ProteinL binds antibodies predominantly through kappa light chain interactionswithout interfering with the antigen-binding site. Chateau et al. (1993)“On the interaction between Protein L and immunoglobulins of variousmammalian species” Scandinavian J. Immunol., 37: 399-405. Protein L hasbeen shown to bind strongly to human kappa light chain subclasses I, IIIand IV and to mouse kappa chain subclasses I. Protein L can be used topurify relevant kappa chain-bearing antibodies of all classes (IgG, IgM,IgA, IgD, and IgE) from a wide variety of species, including human,mouse, rat, and rabbit. Protein L can also be used for the affinitypurification of scFv and Fab antibody fragments containing suitablekappa light chains. Protein L-based reagents is commercially availablefrom Actigen, Inc., Cambridgem, England. Actigen can provide a line ofrecombinant Protein products, including agarose conjugates for affinitypurification and immobilized forms of recombinant Protein L and A fusionprotein which contains four protein A antibody-binding domains and fourprotein L kappa-binding domains.

Other affinity matrix may also be used, including those that exploitpeptidomimetic ligands, anti-immunoglobulins, mannan binding protein,and the relevant antigen. Peptidomimetic ligands resemble peptides butthey do not correspond to natural peptides. Many of Peptidomimeticligands contain unnatural or chemically modified amino acids. Forexample, peptidomimetic ligands designed for the affinity purificationof antibodies of the IGA and IgE classes are commercially available fromTecnogen, Piana di Monte Verna, Italy. Mannan binding protein (MBP) is amannose- and N-acetylglucosamine-specific lectin found in mammaliansera. This lectin binds IgM. The MBP-agarose support for thepurification IgM is commercially available from Pierce.

Immunomagnetic methods that combine an affinity reagent (e.g. protein Aor an anti-immunoglobulin) with the ease of separation conferred byparamagnetic beads may be used for purifying the antibody produced.Magnetic beads coated with Protein or relevant secondary antibody may becommercially available from Dynal, Inc., NY; Bangs Laboratories,Fishers, Ind.; and Cortex Biochem Inc., San Leandro, Calif.

Direct expression and purification of the selected antibody in yeast isadvantageous in various aspects. As a eukaryotic organism, yeast is moreof an ideal system for expressing human proteins than bacteria or otherlower organisms. It is more likely that yeast will make the scFv, Fab,or fully assembled antibody in a correct conformation (foldedcorrectly), and will add post-translation modifications such as correctdisulfide bond(s) and glycosylations.

Yeast has been explored for expressing many human proteins in the past.Many human proteins have been successfully produced from the yeast, suchas human serum albumin (Kang, H. A. et al. (2000) Appl. Microbiol.Biotechnol. 53: 578-582) and human telomerase protein and RNA complex(Bachand, F., et al. (2000) RNA 6: 778-784).

Yeast has fully characterized secretion pathways. The genetics andbiochemistry of many if not all genes that regulate the pathways havebeen identified. Knowledge of these pathways should aid in the design ofexpression vectors and procedures for isolation and purification ofantibody expressed in the yeast.

Moreover, yeast has very few secreted proteases. This should keep thesecreted recombinant protein quite stable. In addition, since yeast doesnot secrete many other and/or toxic proteins, the supernatant should berelatively uncontaminated. Therefore, purification of recombinantprotein from yeast supernatant should be simple, efficient andeconomical.

Additionally, simple and reliable methods have been developed forisolating proteins from yeast cells. Cid, V. J. et al. (1998) “Amutation in the Rho&GAP-encoding gene BEM2 of Saccharomyces cerevisiaeaffects morphogenesis and cell wall functionality” Microbiol. 144:25-36. Although yeast has a relatively thick cell wall that is notpresent in either bacterial or mammalian cells, the yeast cells canstill keep the yeast strain growing with the yeast cell wall stripedfrom the cells. By growing the yeast strain in yeast cells without thecell wall, secretion and purification of recombinant human antibody maybe made more feasible and efficient.

By using yeast as host system for expression, a streamlined process canbe established to produce recombinant antibodies in fully assembled andpurified form. This may save tremendous time and efforts as compared tousing any other systems such as humanization of antibody in vitro andproduction of fully human antibody in transgenic animals.

In summary, the compositions, kits and methods provided by the presentinvention should be very useful for selecting proteins such as humanantibodies with high affinity and specificity against a wide variety oftargets including, but not limited to, soluble proteins (e.g. growthfactors, cytokines and chemokines), membrane-bound proteins (e.g. cellsurface receptors), and viral antigens. The whole process of libraryconstruction, functional screening and expression of highly diverserepertoire of human antibodies can be streamlined, and efficiently andeconomically performed in yeast in a high throughput and automatedmanner. The selected proteins can have a wide variety of applications.For example, they can be used in therapeutics and diagnosis of diseasesincluding, but not limited to, autoimmune diseases, cancer, transplantrejection, infectious diseases and inflammation.

EXAMPLE Example 1 Construction of Expression Vectors Containing HumanSingle-Chain Antibody scFv Library Using Homologous Recombination InVivo

The following illustrates examples of how to use general homologousrecombination as an efficient way of constructing recombinant human scFvlibrary. The coding sequence of each member of the scFV library includesa heavy-chain variable region V_(H) and a light-chain variable regionV_(L) derived from a library of human antibody repertoire. The scFvlibrary is fused with a two-hybrid system activation domain (AD) to forma two-hybrid expression vector in the yeast.

1) Isolation of Human scFv cDNA Gene Pool

A complex human scFv cDNA gene pool is generated by using the methoddescribed in Sambrook, J., et al. (1989) Molecular Cloning: a laboratorymanual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; andAusubel, F. M. et al. (1995) Current Protocols in Molecular Biology”John Wiley & Sons, NY.

Briefly, total RNA is isolated from the white cells (mainly B cells)contained in peripheral blood supplied by un-immunized humans. Bloodsample at 500 ml, which contains approximately 10⁸ B-lymphocytes, areobtained from healthy donors from Stanford Hospital Blood Center. Thewhite blood cells are separated on Ficoll and RNA is isolated by amodified method. Sambrook, J., et al. (1989), supra; and Zhu, L. et al.(1997) “Yeast Gal 4 activation domain fusion expression libraries” in“The Yeast Two-Hybrid System”, S. Fields and P. Bartel, Ed., OxfordUniversity Press, pages 73-98.

If starting from tissue, RNA is first isolated using standardprocedures. Ramirez, F. et al. (1975) “Changes in globin messenger rNAcontent during erythroid cell differentiation” J. Biol. Chem.250:6054-6058; and Sambrook, J., et al. (1989), supra. First strand cDNAsynthesis is performed using the method of Marks et al. in which a setof heavy and light chain cDNA primers are designed to anneal to theconstant regions for priming the synthesis of cDNA of heavy chain andlight chains (both kappa and lambda) antibody genes in separate tubes.Marks et al. (1991) Eur. J. Immunol. 21: 985-991.

Alternatively, human spleen or leukocyte cDNA can be purchased directlyfrom commercial source, such as Clontech, Palo Alto, Calif.

2) PCR Amplification of Heavy and Light Chain Genes

The coding sequences of human heavy and light chain genes are amplifiedfrom the cDNA library generated above by using a method described bySblattero and Bradbury (1998) Immunotechnology 3: 271-278. This methodallows almost 100% coverage of all human V_(H), Vλ and Vκ genes from theknown Ig gene database. Specifically, cDNA pool from human spleen isused (human spleen Marathon-Ready cDNA, Cat.#7412-1, Clontech, PaloAlto, Calif.). Alternatively, cDNA pool from human leukocytes can alsobe used (human leukocyte Marathon-Ready cDNA, Cat.#7406-1. Clontech,Palo Alto, Calif.).

The V_(H), Vλ and Vκ genes are amplified separately by PCR using a setof mixed 5′ and 3′ primers for each class. The 5′ and 3′ primers forV_(H), Vλ and Vκ genes also contain flanking sequences at both endshomologous to a cloning library vector, pACT2 (catalog No. K1604-A,Clontech, Palo Alto, Calif.; Harper et al (1993) “The p21Cdk-interacting protein Cip1 is a protein inhibitor of G1cyclin-dependent kinase” Cell 75: 805-816). Each flanking sequence addedto the primary PCR product is 60 bp in length. The design of theflanking sequence of primer is such that the reading frame of the V_(H)and V_(L) fragments are conserved with upstream GAL 4 reading frame thatis encoded by the cloning vector. Depending on the cloning vector usedin the next step, additional features such as epitope tags (fordetection and purification) and unique restriction enzyme recognitionsites (for subcloning) can also be integrated at this step by primerdesign.

The amplified V_(H), Vλ and Vκ genes are cloned sequentially into thepACT2 cloning vector in yeast via homologous recombination following theschemed depicted in FIG. 2.

Table 2 lists the primer sets used in combination in this method ofsequential homologous recombination. Each of the V_(H) 5′-primers (backprimers), VH1b-VH7b, contains a 60-bp flanking sequence (underlined)homologous to the upstream of the MCS site of pACT2. Each of the V_(H)3′-primers (forward primers), VH1f-VH6f, contains a 60-bp flankingsequence encoding a linker peptide sequence (G₄S)₄ (underlined) [SEQ IDNO: 75]. The V_(H) 5′-primers and the V_(H) 3′-primers are used incombination to amplify the heavy-chain regions of the human antibodygene pool from the cDNA library. The resulting PCR fragments can be usedfor subsequent insertion into the pACT2 vector via the first-stephomologous recombination.

Each of the Vλ (or Vκ) 5′-primers (back primers), Vλ1b-Vλ9b (or Vκ1b-Vκ4b) contains the 60-bp flanking sequence encoding a linker peptidesequence (G₄S)₄ (underlined). Each of the Vλ (or Vκ) 3′-primers (forwardprimers), Vλ1f and Vλ2f (or Vκ1f-Vκ4f), contains a 60-bp flankingsequence (underlined) homologous to the downstream of the MCS site ofpACT2. The Vλ (or Vκ) 5′-primers and the Vλ or Vκ 3′-primers are used incombination to amplify the light-chain regions of the human antibodygene pool from the cDNA library. The resulting PCR fragments can be usedfor subsequent insertion into the pACT2 vector via second-stephomologous recombination.

The PCR reaction is done in the volume of 50 ul containing 5 ul of thecDNA synthesized from step 2, 20 pmol concentration of the mixed 5′ and3′ primers, 250 uM dNTPs, 10 mM KCl, 10 mM (NH4)₂SO₄, 20 mM Tris.HCl (pH8.8), 2.0 mM MgCl2, 100 mg/ml BSA, and 1 ul (1 unit) of KlenTaq DNApolymerase (New England Biolabs, MA). The reaction mixture is subjectedto 30 cycles of amplification using a Perkin-Elmer thermal cycler. Thecycle is 94° C. for 1 min (denaturation), 57° C. for 1 min (annealing),and 72° C. for 2.5 min (extension). Vλ and Vκ chain PCR products arepooled together at this stage. The PCR products are checked byelectrophoresis and purified from 1.0% agarose gel using Qiax affinitymatrix (Qiagen, Calif.) and resuspended in 25 ul of H₂O.

3) Alternative design: PCR Assembly of V_(H) and V_(L) into a SingleFragment

V_(H) and V_(L) (Vλ and Vκ) gene fragments isolated and amplified abovecan be assembled into a single fragment by the overlapping PCR primingmethod. This is a step utilizing the linker region sequence added to the3′ end of V_(H) and 5′ of V_(L) fragments. A typical linker region is atandem repeat of 4 amino acids (G₄S)₃₋₄, and the linker used in thisexample is (G₄S)₄ [SEQ ID NO: 75]. Each single V_(H) or V_(L) PCRproduct is about 420-480 bp, whereas the combined V_(H) and V_(L)fragment, is about 800-850 bp. The V_(H) and V_(L) (Vλ and Vκ) genefragments amplified and isolated as described above are assembled in PCRreactions via the homologous linker sequences shared between the3′-primers of the V_(H) gene and the 5′-primers of the V_(L) gene (Vλand Vκ). The result PCR fragment combining V_(H) and V_(L) linked by the(G₄S)₄ linker L is referred as VH-L-VL. Conditions used for the PCRassembly is same the PCR for amplifying the V_(H) and V_(L) genesseparately as described above, except that the cycle number is 20.

The PCR assembled product containing both V_(H) and V_(L) gene fragmentsare analyzed by agarose electrophoresis, and are purified from agarosegel by Qiax method (Qiagen, C A).

4) Cloning of Heavy- and Light-Chain Fv Fragments into a Two-Hybrid ADVector by Homologous Recombination in Yeast

The PCR fragments of V_(H) and V_(L) cDNA gene pool generated above arecloned into a two-hybrid vector containing an activation domain (AD) byhomologous recombination in one step by using the combined singlefragments VH-L-VL generated above.

The two-hybrid vector containing an AD domain, pACT2 is purchased fromClontech, Palo Alto, Calif. In this example, 10 μg of pACT2 islinearized with restriction enzymes digestion in the multiple cloningsites (MCS). This is done in 20 ul volume containing the followingreagents: 10 μg of vector DNA, 1-2 ul of each restriction enzyme BamH Iand Xho I, 2 ul of 10× buffer. Digestion is carried out at 37° C.overnight. The completion of the enzyme digestion is checked byelectrophoresis. No further modification or purification of linearizedvector is necessary.

The linearized vector DNA (10 μg) is mixed with equal amount of the PCRamplified VH-L-VL fragments (about 5-10 molar excess of the insertfragment) in a single fragment as described in section 3). Thelinearized vector DNA and the PCR fragment are co-transformed intocompetent yeast strain Y187 (α mating type, from Clontech).

Transformation is performed as the following. Yeast competent cells areprepared by LiAc protocol (Gietz et al. (1992) “Improved method for highefficiency transformation of intact yeast cells” Nucleic Acids Res. 20:1425), or obtained from a commercial source (Life Technology Inc., Md.).Minimum yeast competency of 10⁶ transformant/ug DNA may be required forlibrary construction. Yeast competent cells derived from 1 liter cultureof OD600=0.2 are used for each transformation in 50 ml conical bottomtubes. Yeast cells are thawed at 4° C., washed with de-ionized water andresuspended in 8 ml of 1×TE/LiAc (1×TE/LiAc is made up of 40%polyethylene glycol 4000, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5, and 0.1 Mlithium acetate). The mixture of DNA containing the linearized vectorand PCR amplified inserts with extended ends is added to the tube andvortexed to mix. The tube is incubated at 30° C. for 30 min, withshaking (200 rpm). DMSO (Dimethyl sulfoxide, 700 ul) is added into thetube and mixed gently. The cells in the tube are heat shocked at 42° C.in a water bath for 15 minutes with occasional swirl. After the heatshock, the cells are pelleted by a brief centrifugation at 4° C. andwashed one or two time with water. The cells are resuspended in 1.5 mlof 1×TBE buffer.

Yeast cells are plated into plates made up of selection medium. For Y187strain of yeast, the SD/-Leu medium is used. Harper et al. (1993),supra. The library scale transformation requires approximately 100 largeplates of 150 mm in diameter. Y187 transformed with either linearizedvector without insert DNA fragment or vise versa is also plated onto thesame selection plates as controls. Y187 transformed with unlinearizedvector pACT2 is used as transformation efficiency control and is platedwith series dilutions. The plates are incubated bottom up at 30° C. for3 days or more. Colony number is examined and recorded. If the yeastcontrol transformation with unlinearized pACT2 yields at least 1 milliontransformants, as expected, 10 millions of single chain libraryrecombinant clones are expected to obtain from each such transformation.Any control transformation with either the linearized vector or insertDNA fragment alone is expected to yield only {fraction (1/10)} or lessnumber of colonies as compared with the combined vector/inserttransformation. This single step of transformation is repeated until 100million or more independent clones are obtained.

For the separate PCR fragments of V_(H) and V_(L) as described insection 2), the V_(H) and V_(L) fragments are inserted into a modifiedpACT2 cloning vector separately following the scheme depicted in FIG. 2.This is achieved by sequential transformations consisting of twoindependent events of homologous recombination in vivo.

The original pACT2 plasmid is modified by oligonucleotide-directedmutation. FIG. 13 illustrated the plasimid map of pACT2 and the methodof modification. As illustrated in FIG. 13, an oligonucleotidecontaining the linker sequence encoding the linker peptide (G₄S)₄ and afew unique restriction sites (e.g. BssH I and Pac I) is inserteddownstream from the stop codon of the AD domain in pACT2. Table 3 listssense [SEQ ID NO: 46] and antisense strands [SEQ ID NO: 47] of theoligonucleotides used to modify pACT2.

The sense and antisense strands listed in Table 3 are annealed andphosphorylated by T4 DNA kinase (New England Biolabs, MA). The annealeddouble-stranded DNA fragment contains these features in this order: acohesive end of Xho I (functional after ligation), the (G₄S)₄ linkersequence, BssH 2 site, Pac I site, and another cohesive end of Xho I(not functional after ligation). As illustrated in FIG. 13, the annealedfragment are then ligated to a Xho I-digested pACT2 which has beendephosphorylated by calf intestinal alkaline phosphatase. After theorientation of the inserted fragment in the new vector (designatedpACT2-GS) is confirmed, the two-step homologous recombination isperformed.

The modified yeast cloning vector, pACT2-GS, is digested with BamH I andXho I. The V_(H) and V_(L) inserts in separate PCR fragments asdescribed in section 2), are incorporated sequentially into thelinearized pACT2-GS vector in Y187 yeast cells via homologousrecombination.

The PCR fragments of V_(H) are inserted into the BamH I and XhoI-linearized pACT2-GS vector downstream of the AD domain, but upstreamof the (G₄S)₄ linker sequence via homologous recombination. Thistransformation is preferred to yield at least 1 million independentclones.

After this first round of homologous recombination, the yeast cells arepooled and the plasmid DNA with the V_(H) insert is recovered andtransformed into bacterial cells for plasmid preparation. Competent E.coli strain KC8, either chemical or electrical competent (available fromClontech, Palo Alto, Calif., Cat #C2004-1 or #C2023-1, respectively) isused for a convenient amino acid complementation as selection for pACT2with its leucine marker. The plasmid are prepared from KC8 cells inlarge scales for a second round yeast transformation by an independenthomologous recombination.

The pACT2 vector having the V_(H) insert is linearized by Pac I,downstream from the (G₄S)₄ linker sequence. The resulting PacI-linearized vector (10 μg) and the PCR-fragments of V_(L) aretransformed into Y187 yeast cells where the V_(L) fragments are insertedinto the vector via a second homologous recombination. The transformantsare plated again in SD/-Leu selection plates. Such single transformationis preferred to yield 100 million or more independent clones. Theseclones are double homologous recombined library clones with both humanV_(H) and V_(L) chains.

The yeast library recombinant colonies generated as described above arescraped from the final culture plates after growing for 5-7 days. Themajority of the yeasts are mixed with 50% (volume) of glycerol andstored at −80° C. for future library screening use. A small fraction ofthe yeast clones are subjected to the following quality analyses:

-   a. Percentage of recombinant clones: PCR amplification of the insert    (either the VH-L-VL insert in a single PCR fragment, or V_(H) and    V_(L) in separate PCR fragments, depending on the procedure used    in 2) OR 3) above) directly from yeast with a primer pair matched    with flanking vector sequences (e.g., Long PCR primer pair for AD    vectors supplied by Clontech) should reveal how many clones are    recombinant. Since our design of extended homologous regions for    recombination between the insert and cloning vector is sufficient    long (about 60 bp), a high percentage of recombinant clone (higher    than 95%) should be expected. Libraries with minimum of 90%    recombinant clones are preferably to be saved for screening use.-   b. Insert size: The same PCR amplification of selected clones should    reveal the insert size. Although a small fraction of the library may    contain double or other forms of multiple inserts, the majority    (>95%) should have single insert with expected size.-   c. Fingerprinting verification of sequence diversity: PCR    amplification product with the correct size is fingerprinted with    frequent digesting restriction enzymes, such as Bst NI or any other    3-4 base cutters. From the agarose gel electrophoresis pattern, one    can determine whether clones analyzed are of the same identity or of    the distinct or diversified identity. The PCR products can also be    sequenced directly. This will reveal the identity of inserts and the    fidelity of the cloning procedure, and will prove the independence    and diversity of the clones. If 100 clones are sequenced, it should    be expected that only small fraction (<5%) of clones will have    multiple isolates.

Example 2 Construction of Human scFv Library by Using CRE/loxP-MediatedRecombination In Vivo

In this example, the construction of a highly complex and diversecombinatorial repertoire in yeast using V-region gene segments asbuilding blocks is described.

First, a special type of human scFv library is generated in yeast by thestandard homologous recombination procedure underlined in Example 1.This library is consisting of 10⁷ or more of highly diverse and complexV-region gene repertoire derived from heavy chain and light chainorigin. One pool (e.g., V_(L) or light chain gene segment) is flanked onboth sides by two non-identical lox P sites. The loxP sites are designedinto the primer sequences used in one of the PCR amplification steps.Examples of the loxP sites are listed in Table 1.

Specifically, two nonidentical loxP sites, loxP1 [SEQ ID NO: 4] andloxP2 [SEQ ID NO: 5] (Table 1), are incorporated into the PCR primersfor amplifying the V_(H) and V_(L) gene segments from the cDNA libraryas described in Example 1, Section 2).

Table 4 lists the primer sets used in combination for amplifying theV_(H) and V_(L) gene segments from the cDNA library. Each of the V_(H)5′-primers (back primers), VH1b-VH7b, contains a 60-bp flanking sequence(underlined) homologous to the upstream of the MCS site of pACT2. Theseprimers are the same as those used for amplifying V_(H) gene segmentswithout incorporating the loxP sites.

Each of the V_(H) 3′-primers (forward primers), VH1′f-VH6′f, contains a63-bp flanking sequence (underlined). The V_(H) 5′-primers and the V_(H)3′-primers are used in combination to amplify the heavy-chain regions ofthe human antibody gene pool from the cDNA library. (Note: The resultingPCR fragments can be used for subsequent insertion into the pACT2 vectorvia the first-step homologous recombination as illustrated in FIG. 2.)

Each of the Vλ (or Vκ) 5′-primers (back primers), Vλ1′b-Vλ9′b (orVκ1′b-Vκ4′b), contains a 63-bp flanking sequence that is complementaryto the 63-bp flanking sequence of the V_(H) 3′-primers and comprisesthese sequences in 5′ to 3′ order: a (G₄S) coding sequence, a loxP1site, and a G₃S coding sequence (underlined).

Each of the Vλ (or Vκ) 3′-primers (forward primers), Vλ1′f and Vλ2′f (orVκ1′f-Vκ4′f), contains a 30-bp flanking sequence (underlined) that is apartial loxP2 site. The Vλ (or Vκ) 5′-primers and the Vλ or Vκ3′-primers are used in combination to amplify the light-chain regions ofthe human antibody gene pool from the cDNA library. The resulting PCRfragments are further amplified by using a new 3′ primer (listed as theVλ/Vκf primer in Table 4) in a secondary PCR to incorporate the fullloxP M2 site and a sequence homologous to the downstream of the MCS siteof pACT2. This design allows amplification of the VL gene segments byusing shorter primers. (Note: The amplified VL segments can be used forsubsequent insertion into the pACT2 vector via the second-stephomologous recombination as illustrated in FIG. 2).

The PCR reaction is done in the volume of 50 ul containing 5 ul of thecDNA synthesized from step 2, 20 pmol concentration of the mixed 5′ and3′ primers, 250 uM dNTPs, 10 mM KCl, 10 mM (NH4)₂SO₄, 20 mM Tris.HCl (pH8.8), 2.0 mM MgCl2, 100 mg/ml BSA, and 1 ul (1 unit) of KlenTaq DNApolymerase (New England Biolabs, MA). The reaction mixture is subjectedto 30 cycles of amplification using a Perkin-Elmer thermal cycler. Thecycle is 94° C. for 1 min (denaturation), 57° C. for 1 min (annealing),and 72° C. for 2.5 min (extension). Vk and VK chain PCR products arepooled together at this stage. The PCR products are checked byelectrophoresis and purified from 1.0% agarose gel using Qiax affinitymatrix (Qiagen, C A) and resuspended in 25 ul of H₂O.

V_(H) and V_(L) (Vλ and Vκ) gene fragments isolated and amplified aboveare assembled into a single fragment by the overlapping PCR primingmethod as illustrated in FIG. 3. This is a step utilizing the linkersequence added to the 3′ end of V_(H) and 5′ of V_(L) fragments. In thiscase, the linker sequence contains a (G₄S) coding sequence a loxP1 site,and a G₃S coding sequence.

Each single V_(H) or V_(L) PCR product is about about 420-480 bp,whereas the combined V_(H) and V_(L) fragment is about 800-850 bp. TheV_(H) and V_(L) (Vλ and Vκ) gene fragments amplified and isolated asdescribed above are assembled in PCR reactions via the homologous linkersequences shared between the 3′-primers of the V_(H) gene and the5′-primers of the V_(L) gene (Vλ and Vκ). The resulting PCR fragmentcombining V_(H) and V_(L), wherein V_(L) is flanked by loxP1 and loxP2sites, is referred as VH-loxP1-VL-loxP2. Conditions used for the PCRassembly is the same PCR for amplifying the V_(H) and V_(L) genesseparately as described above, except that the cycle number is 20.

The PCR assembled product containing both V_(H) and V_(L) gene fragments(the VH-loxP1-VL-loxP2 fragments) are analyzed by agaroseelectrophoresis, and are purified from agarose gel by Qiax method(Qiagen, C A).

The VH-loxP1-VL-loxP2 fragments are inserted into the pACT2 vector viahomologous recombination in vivo following the general scheme depictedin FIG. 3. The procedure is the same as the one used for homologousrecombination between pACT2 and the VH-L-VL fragments described insection Example 1, Section 4).

After a library is generated and 10⁷ of independent clones areaccumulated, this entire library of pACT2 plasmids containing theVH-loxP1-VL-loxP2 sequences is isolated from pooled yeast clones andthen transformed into E. coli strain KC8 through the shared leucinenutritional marker complementation. Bacterial strain KC8 carries hisB,leuB, and trpc mutations. These mutations exhibit amino acids deficiencyand can be complemented by the corresponding genes from yeasts. SeeYeast Protocol Handbook, Clontech, PT3024-1, page 33.

Isolation of the plasmid pool from the yeast cells is done using thelyticase protocol. Guthrie and Fink (1991) “Guide to yeast genetics andmolecular biology” in Methods in Enzymology (Academic Press, San Diego)194: 1-932. Briefly, the library clones grown in selection plates arescraped out and resuspended in 1×TE. A freshly made 5 units/ul lyticase(Sigma, St. Luis, Miss.) solution is added to the yeast suspension at1:5 v:v ratio and the mixture is incubated at 37 C for 60 min withoccasional swirling. Using dissect microscope to check the degree ofyeast wall digestion. If the yeast wall is completely digested, yeastcells will burst immediately in water or low salt solution. When thedigestion is completed, add 20% of SDS at 1:5 v:v ratio and continue toincubate for a few minutes. The yeast suspension is subject to severalfreeze-thaw cycles by placing the tube in dried ice and water bath (37°C.). At this stage, the suspension is passed through a DNA fractionationcolumn, e.g., CHROMA Spin 1000 from Clontech, Palo Alto, Calif. Thecolumn cleans up the cell debris and other components, only lettingplasmid DNA to pass through. The plasmid DNA can be collected by washingthe column with 1×TE.

This collection of yeast plasmid DNA is then used for transformationinto E. coli stain KC8 (Chemical or electrical competent KC8 cells canbe ordered from Clontech, Cat #C2004-1 or #C2023-1). In either case,when the bacterial cell is transformed with the plasmid pool isolatedfrom yeast, selection plates made of M9 minimum medium is used forplating the bacteria. Because the shared nutritional selection marker,leucine deficiency, is present in both the yeast strain and thebacterial strain, the plasmid which carries the dominant marker, can berescued from the bacteria. Finally, the KC8 cells are let to grow and alarge-scale DNA isolation from KC8 is done for DNA pools which aresubsequently mixed.

This pooled DNA source is then re-introduced into yeast host strain Y187using conventional single plasmid transformation protocol. The conditionof this transformation is set to enrich for multiple plasmid entry intoevery single yeast cells. Yeast can take multiple plasmids asdemonstrated by the two-hybrid system design where both AD and BDplasmids are co-existing in the same yeast host cells. A normalsmall-scale yeast transformation with 1 ug level of DNA will give riseto an average of yeast transformants with 30-50 copies of plasmid.

The multiple plasmid entry into yeast is maximized by using higherDNA-yeast ratio in the transformation step. The yeast cells are alsopre-transformed with a plasmid that inducibly expresses CRE recombinase.The inducible expression of CRE in the yeast strain causes theCRE-mediated site-specific recombination at the Lox P sites that flankeach light chain gene fragment. Therefore, while yeast is allowed togrow and the plasmids in the yeast cells are making additional copies,shuffling of the light chain gene segment VL (Vλ and Vκ) should occurinside of yeast cells. This process of CRE/loxP-mediated chain shufflingis illustrated in FIG. 4A.

Assuming this shuffling is totally random and complete with the entirepool, the total number of combination of heavy chain and light chainwithin the yeast cells will be increased exponentially. Thus, a librarywith at least 10⁹ of recombinant clones can be generated. Theoretically,the complexity of the library can reach 10¹⁴ if the starting library hasa complexity of 10⁷.

This recombination in yeast should not require any marker selection. TheCRE/loxP recombination should occur irrespective of with selection orwithout selection. The key to success in this example is multiple entryof plasmid into the yeast cells which is a norm in the yeasttransformation. This mode of multiple plasmid entry is tested by usingdifferent color GFP variant plasmids. For example, plasmids harboringGFP (encodes green fluorescent protein) or YFP (a mutant form of GFPthat encodes yellow fluorescent protein) are mixed at 1:1 ratio and usedfor yeast transformation. These plasmids should have no difference intheir structures in terms of selection marker or plasmid compositionexcept for the expression cassette (either GFP or YFP). The codingregions of these two fluorescent proteins are of the same length andonly differ from each other in very few amino acids (S65G, V68A, S72Aand T203Y) Miller D. M., (1999) Biotechniques 26: 914-918. Theseplasmids resemble the library of expression vectors that carry the humanantibody coding variable regions in that all antibody molecules areessentially of the same length and differ from each other only in asmall number of amino acid compositions, most in the hypo-variableregions. If yeast takes multiple fluorescent protein plasmids, certainfraction of yeast transformed should show a combined color spectrum.Some colonies will show a mosaic phenotype. This test also allows foroptimization of the condition for multiple plasmid transformation.

Example 3 Construction of Human scFv Library of Very High Complexity byUsing CRE/loxP-Mediated Recombination In Vivo-Second Design

An alternative method to the method described in Example 2 forconstruction of human scFv library using CRE/loxP-mediated recombinationis to use a “forced” multiple transformation. In this design, twostarting human scFv libraries containing human heavy and light chaingene segments are generated separately in two vectors with differentselection markers (e.g., Leu 2 and Ade 2, respectively). By selection ofboth markers will ensure that every yeast cell have both types oflibrary clones (each may have multiple but variable number of copies).The activation or expression of Cre combinase in the yeast should allowthe CRE/loxP-mediated recombination as illustrated in FIG. 4B.

Two special human scFv libraries are generated in yeast via homologousrecombination by using the procedures described in Example 2. The twolibraries are otherwise the same in terms of their source RNA,amplification, and the cloning procedures. The only difference is thecloning vector used. One library contains the human scFv library carriedby an unmodified pACT2 with Leu 2 as a yeast selection marker, while theother contains the human scFv library carried by a modified pACT2 withAde 2 as a yeast selection marker. Each library includes 10⁷ or more ofhighly diverse and complex V-region gene repertoire derived from heavychain and light chain origins of human antibody. The VH-loxP1-VL-loxP2PCR fragments generated in Example 2 are inserted into the linearizedpACT2 vector with Leu 2 and the linearized pACT2 vector with Ade 2respectively, via homologous recombination in yeast. The results in twolibrary of scFv carried by two different pACT2 vectors with differentselection markers.

After 10⁷ of independent clones are accumulated in each of these twolibraries, this entire library DNA is isolated from pooled yeast libraryclones and then transformed into bacterial strain KC8 throughbacteria-yeast leucine nutritional marker complementation. Theprocedures are similar to those described in Example 2. Large-scale DNAisolations from KC8 are done from the two libraries and the two DNApools are kept separately. These two pooled DNA sources areco-transformed into yeast Y187 cells at 1:1 ratio by using conventionalsingle plasmid transformation protocols.

Y187 has the following genotype: Matα, ura3-52, his3-200, ade2-101,trp1-901, leu2-3, 112, gal4Δ, met⁻, gal80Δ,URA3::GAL1_(UAS)-GAL1_(TATA)-lacZ. Harper, et al. (1993) Cell 75:805-816. It allows both types of plasmids to be selected and maintainedby Leucine and Adenine complementation. The condition of thistransformation is similar to the standard plasmid transformation and canbe modified to reach a maximum efficiency of transformation.

The transformants are plated onto SD/-Leu/-Ade medium for selecting bothtypes of library plasmids. Any yeast colonies formed on this doubleselection medium must have transformed by both types of library clones.Each type of the library clone is in multiple copies, usually at 30-50copies per cell. Except for a few particular individual cells, thepairing of the two library clones should be totally random.

Similar to the yeast cells in Example 2, the yeast cell is alsopre-transformed with a plasmid that inducibly expresses CRE recombinase.The inducible expression of CRE recombinase in the yeast strain causesthe Cre-mediated homologous recombination at loxP sites flanking eachlight chain gene fragment.

Therefore, while yeast is allowed to grow and the plasmids in the yeastcells are making additional copies, shuffling of the light chain genesegment V_(L) should occur inside of yeast cells. This process ofCRE/loxP-mediated site specific recombination is illustrated in FIG. 4B.Assuming this shuffling is totally random and is complete with theentire pool, the total number of combination of heavy chain and lightchain within the yeast cells will be increased exponentially. We canthus generate a library with at least 10⁹ of recombinant clones.Theoretically, the complexity of the library can reach 10¹⁴ if thestarting two libraries each has a complexity of 10⁷.

Example 4 Screening of Antibody Single Chain Fv Libraries in Yeast withthe Two-Hybrid System Against Defined Protein Antigens Via MatingBetween Two Yeast Strains

This example describes a procedure used to screen the antibody scFvlibraries generated in the Examples 1, 2 and/or 3. The scFv librariescontaining human V_(H) and V_(L) segments are generated in yeast strainwith an α mating type. This mating type of yeast can be readily matedwith an a type of yeast with simple mating procedure to form diploidyeast cells. Guthrie and Fink (1991) “Guide to yeast genetics andmolecular biology” in Methods in Enzymology (Academic Press, San Diego)194: 1-932. The a-yeast contains the target (probe, or bait) plasmid.

The target plasmid contains a fusion formed between the GAL 4 DNAbinding domain (BD) and any desired target protein that is to be used asa probe to fish out the antibodies as its affinity ligand. When the twotypes of yeast cell mate and form diploid cells, the probe plasmid andthe library clone plasmid also come together in a same cell. Therefore,if a specific antibody scFv clone recognizes and binds to the probeprotein, each of these proteins or protein fragments should bring theirfusion partners (GAL 4 AD and GAL 4 BD) to a close proximity in thepromoter region of reporter(s). Under such a circumstance, thereporter(s) construct built in the yeast cells (the parental a- and/orα-type of haploid cells) should be activated by the active GAL 4proteins. Thus the reporter is expressed and a positive signal in thelibrary screen is detected. Certain reporter(s) are of nutritionalreporter, which allows the yeast to grow on a specific selection mediumplate.

In practice, equal volume of bait-containing yeast strain (a-type) andscFv library-containing yeast stain (α-type) are inoculated intoselection liquid medium and incubated with rigorous shaking at 30° C.for 20 hours. These cultures are then mixed in a single flask andallowed to grow in rich medium 1×YPD (20 g/l Difco peptone, 10 g/l yeastextract, and 2% glucose) for 12-16 additional hours with slow shaking at30° C. Under the rich nutritional culture condition, the two haploidyeast strains encounter and mate to form diploid cells. At the end ofthis mating process, a good fraction—5-10% of the yeast populationpresent in the mating pool will form diploids. Bendixen, C., Gangloff,S., and Rothstein, R. (1994) “A yeast mating-selection scheme fordetection of protein-protein interactions” Nucleic Acids Res. 22:1778-1779.

After mating, the yeast cells are washed with H₂O several times andplated into selection plates by using the SD/-Leu-Trp-His-Adeselections. The first two selections are for selection markers (Leu andTrp) expressed from the vectors and are for retaining both BD and ADvectors in the same yeast cells. The selected cells should be diploidcells, since either haploid cell only expresses one of these markers.The latter two markers are expressed by the reporter from the hoststrains and are for selection of clones that show positive interactionbetween the members of the scFv library and the target protein.

Example 5 Screening of Single Chain Fv Antibody Libraries Against aLibrary of Antigens in a Yeast Two-Hybrid System

For small number of pre-selected probes, the procedure of individualmating screening as described above is sufficient. However, thisprocedure can also be modified to suit for screening against largenumber of targets or probes. The following list describes the potentialprobes that are in large number and may not suitable for individualmating screening:

-   a. A collection of human EST clones, or total library of human EST.    Such EST collection can be ordered from public resource in a library    format with individually clones arrayed in 96-well or 384-well    plates. The EST inserts from the original collection (usually in    bacterial cloning and sequencing vectors) are PCR amplified with    extended homologous sequences at both ends. The EST inserts can be    PCR amplified and additional flanking sequences can be added to both    ends of the ESTs by PCR for mediating homologous recombination in    yeast. Then through the same homologous recombination procedure    describe in Examples 1 and 2, the EST insert can be cloned into the    AD vector. A maximum of three homologous recombination events should    be sufficient for the read-through fusion of each EST with the GAL4    AD. Hua, S. B. et al. (1997) “Minimum length of sequence homology    required for in vitro cloning by homologous recombination in yeast”    Plasmid 38: 91-96.-   b. A collection of certain domain structures, such as zinc finger    protein domains each having 18-20 amino acids. These domain    structures may not be completely random. Synthetic oligonucleotides    with characteristic conserved and random/degenerate residues can be    made to cover most of the rational domain structures;-   c. A completely random peptide library each having 16-20 amino acid    residues. Such a library can also be made by random oligonucleotide    synthesis. Such library has been constructed in an AD vector.    Yang, M. et al. “(1995) “Protein-protein interactions analyzed with    the yeast two-hybrid system” Nucleic Acids Res. 23: 1152-1157. Such    a library of probes can also be built in an BD vector. Each clone of    such library represents a short peptide. The scFv antibody library    (built in AD vector) is screened against this library of probes,    peptide ligands for each scFv antibody can be selected. Such    peptides may have potential applications in rational design and    structural improvement of antigens.

The library of probes are cloned into a DB vector and each is fused withGAL4 DB domain. This library are made as an arrayed clone library bydepositing every clone obtained with BD-probe fusion into a well in 96or 384 well plates. This arrayed format facilitates large scale libraryscreening with machine-aided automation.

Prior to using the library of probes to screen against the scFv library,the library of probes are transformed into yeast a-type of host strainto select out any self-activating clones. This pre-selection is to allowthe yeast harboring only the probe plasmids to grow in a selectionmedium (SD/-Trp-His) and check for activation without the AD matingpartner, the so-called self activation.

Alternatively, the pre-selection is conducted in selection medium withα- or β-galactosidase substrate. Any positive clones will produce acolored reaction and can be easily detected by naked eye or byinstrument. The clone that send out positive signals indicatingactivation of the reporter gene(s) are self-activating clones which areexcluded from the subsequent use as the targets for the scFv library.

The machine-aided automatic screening is performed by using 96- or384-well plates. The target clones of a-strain are sequentiallyinoculated into a plate which is pre-seeded with an arrayed library ofthe scFv library of α-strain. The two haploid yeast strains mate in therich medium and form diploid. The wells sending positive signals ofreporter gene expression are detected. The screening process is similarto the individual target screening against a library in the mixedculture as described in Example 3. The difference in this case is thatclonal mating (a mating between an individual target against anindividual scFv) is performed here to enhance the efficiency when largenumbers of targets and scFv antibodies are involved.

Example 6 Maturation of scFv Primary Isolates by Random Mutagenesis InVitro and Re-Screening In Vivo in a Yeast Two-Hybrid System

The scFv clones isolated from in Examples 1-3 can be of various degreeof affinity. Although high affinity clones may be obtained with a lowmarginal possibility, the majority of the clones may need furthermodification to reach affinity compatible with natural antibodies(dissociation constant at 10⁻⁹ M or lower).

In this example, the sequences of primary scFv clones are mutagenized invitro to incorporate random mutations into the V_(H) and V_(L) regions,thereby creating a secondary library of scFv with increased complexity.Complexity of the secondary library is expected to be at 10⁴ or higher.So the combined diversity of primary and secondary libraries screenedshould be at 10¹⁴-10¹⁸, no less than the natural antibodydiversification through selection/maturation in an animal.

Coding sequences of the V_(H) and V_(L) regions of the selected scFv areamplified from the corresponding scFV clones by PCR. The V_(H) and V_(L)region is resided in the AD vector and is fused with GAL4 AD domain. Apair of PCR primers are used to specifically amplify the V_(H) and V_(L)region out of the vector. The pair of primers are designed to match withthe regions of the cloning vectors that flank the V-regions genes. Theseregions contains sequences for homologous recombination between thecloning vector and the amplified product. The PCR product is predictedto be about 0.8 kB.

This primary PCR product is checked by agarose gel electrophoresis forcorrect size and amount. An aliquot of the primary PCR product is thensubjected to a secondary PCR. This secondary PCR is designed toincorporate mutations into the product under these conditions: highconcentration of Mn²⁺ and over-proportionaly high concentration of onenucleotide substrate in the PCR reaction in the PCR reaction. Mn²⁺ at aconcentration of between 0.4 and 0.6 mM can efficiently cause Taqpolymerase to incorporate mutations into the PCR product. Thismis-incorporation is caused by the malfunction of Taq DNA polymerase.Single nucleotide (e.g., dGTP) at an extra higher concentration than theother 3 essential nucleotides (dATP, dTTP, and dCTP) causes theincorrect incorporation of this high concentration substrate into thetemplate and produce mutations.

Besides the two conditions listed above, other condition may influencethe rate of mis-incorporation of “wrong” nucleotide into the PCRproduct, including the number of PCR cycles, the species of DNApolymerase used, and the length of the template. In this example, apre-made kit is used (Diversity PCR Random Mutagenesis Kit, Cat.#K1830-1, Clontech, Palo Alto, Calif.). This kit contains reagentsnecessary for optimizing the conditions for random mutation by PCR, suchas dNTP Mix and additional dGTP solution, Manganese Sulfate, and controlPCR template and primer mix.

As suggested by the user manual for this kit, the following condition isused for PCR mutagenesis: 640 uM MnSO₄, 200 uM dGTP. Under thiscondition, an average of 8 mutations is expected to be found in every1000 bp, a rate that is sufficient for scFv diversification.

This secondary scFv library is reintroduced into yeast throughhomologous recombination and screened directly in yeast followingsimilar procedures as in the primary screening described in Example 3.This whole process mimics the naturally occurring affinity maturationprocess that higher organisms including human are inherited.

Example 7 Expression and Purification of Fully Assembled HumanAntibodies in Yeast

Through the process of primary screening, affinity maturation, andsecondary screening as described in Examples 1-6, specific scFv humanantibody with high affinity toward a given target antigen are selected.The selected antibody can be expressed directly in yeast.

Using the yeast as expression host has several advantages. First, as aeukaryotic organism, yeast is more of an ideal system for expressinghuman proteins than bacteria or other lower organisms. It is more likelythat yeast will make the scFv, Fab, or fully assembled antibody in acorrect configuration and conformation (with correct protein folding),and will add post-translation modifications such as disulfide bond(s)and glycosylations. Second, yeast has been explored for expressing manyhuman proteins in the past. Many human proteins have been successfullyproduced from the yeast. Third, yeast has fully characterized secretionpathways. The genetics and biochemistry of many if not all genes thatregulate the pathways have been identified. Fourth, yeast has very fewsecreted proteases. This makes the secreted recombinant protein quitestable. In addition, yeast does not secrete many other proteins, ortoxic substance such as PLS. So the supernatant is relativelyuncontaminated. Therefore, purification of recombinant protein fromyeast supernatant is simple and desirable.

By using yeast as host system for expression, a streamlined process canbe established to produce recombinant antibodies in fully assembled andpurified form. This should save time and efforts as compared to usingother systems involving animals.

The V_(H) and V_(L) regions of the selected scFv are amplified from thecorresponding clones with primers that simultaneously adding sufficienthomologous recombination sequences to the PCR product. These PCRproducts are then be introduced into a yeast strain together with alinearized expression vector. Through homologous recombination, a newcircle vector are generated which includes the V_(H) and V_(L) regionslinked to the desired promoter upstream and stop codons andtranscription termination signal downstream. A secretion signal is alsoadded in the 5′ end of the V_(H) and V_(L) segments, so the recombinantprotein can be expressed as secreted form.

A few commercially available vectors offer the secretion signal. In thisexample, the PCR fragments of scFv sequences can be cloned into thistype of vector for simple final purification. The expression vectorincludes either a constitutive expression promoter such as ADH1(Ruohonen, Aalto, and Keranen (1995) “Modification of the ADH1 promoterof Saccharomyces cerevisiae for efficient production of heterologousproteins” Journal of Biotechnology 39: 193-203), or an inducibleexpression promoter, such as Gal1 (Flick and Johnston (1990) “Twosystems of glucose repression of the GAL1 promoter in Saccharomycescerevisiae” Mol. Cell Biol. 10: 4757-4769), or GCN4 (Mimran, et al.(2000) Biotechniques, 28: 552-560). The GCN4 inducible promoter ispreferred because the induction can be easily achieved by adding 3-ATinto the yeast culture medium. The scFv antibody fragments thusgenerated will be useful for assessment of affinity and specificity intraditional settings, such as ELISA, western, or immune staining. Ifthey are of good affinity and specificity, they can be used either asbuilding blocks in Fab expression vectors, or can be further assembledwith the constant region for full length antibody expression (seebelow).

The yeast strain to be used for expression can be of any standard strainwith a nutritional selection marker. The marker used for expression inthis example is different from that of the AD vector. This will help toavoid potential carryover problem.

Moreover, fully assembled human antibodies can also be expressed inyeast in secreted form by taking advantage of the fact that yeast cantake and maintain multiple copies of plasmid of the same replicationorigin. This has been successfully used in the two-hybrid system designwhere the BD and AD vectors are identical in backbone structure exceptthe selection markers are distinct. So in this example, the heavy chaingene and light chain gene are co-expressed by two different vectors.Thus, a fully functional antibody protein with two heavy chains and twolight chains can be assembled in the yeast and secreted into the medium.This step not only confers upon the final product higher affinity (oravidity) and stability but also renders the purification of the secretedproduct much easier. The same approach can be used for assembling theFab fragments in the yeast.

The scFv with a constant region, Fab, or fully assembled antibody can bepurified using Protein A, Protein L, or Protein G as affinity matrix.These proteins of bacterial origin are naturally occurring high affinityligands for most classes of Antibody. They are commercially availableand have been used widely in small and large-scale antibodypurification.

Described below in detail is an example for expression of a secretedscFv fragment in yeast. This approach can be easily adapted forexpression of Fab or full-length antibody (e.g., Ig G).

The plasmid pGES426 (Mimran, Marbach, and Engelberg (2000)Biotechniques, 28:552-560) is used as a yeast expression vector. Thisvector contains a backbone derived from pBluescript, 2μ yeastreplication origin for high copy plasmid maintenance, and full lengthGCN4 upstream regulatory sequence (1067 pb) that offers the highestefficiency of expression of heterologous gene. The experiment resultsusing human serum albumin as testing gene showed that this full-lengthupstream sequence is required for the highest level of 3-AT induction. Ayeast secretion signal sequence such as signal sequence of Suc 2 whichencodes invertase gene, Kaizer, C. A. and Botstein, D. 1986, Mol CellBiol. 6: 2382-2391) is cloned into this vector upstream from the uniqueBamH I insert cloning site. The sequence of Suc 2 [SEQ ID NO: 74] isATGCTTTTGC MGCTTTCCT TTTCCTTTTG GCTGGTTTTG CAGCCAAAAT ATCTGCATCA ATG.

The BamH I site is purposely reserved only in the 3′ end of thesecretion signal sequence. A scFv fragment that is obtained throughyeast two-hybrid screening and with desired specificity against a givenantigen probe, is PCR amplified from the original two-hybrid AD-scFvfusion library vector. The PCR primer is designed to amplify the scFvregion sequence and contains an additional translation initiation codonATG at the 5′ end, and a translation stop codon TAA at the 3′ end. Inaddition, a BamH I site is also incorporated into each of the PCRprimers. After PCR amplification using a high-fidelity DNA polymerase(e.g. KlenTaq, Barnes, W. M. (1994) “PCR amplification of up to 35-kbDNA with high fidelity and high yield from λ bacteriophage templates”Proc. Natl. Acad. Sci. USA 91: 2216-2220), the amplified fragment isdigested with BamH I. This fragment is further treated with kinase toadd phosphoryl group to the 5′ ends.

Meanwhile, the vector is also digested with BamH I and dephosphorylatedwith a phosphotase. After that, the scFv fragment is ligated with thelinearized expression vector, and a recombinant clone with correctorientation is isolated. This recombinant clone is then introduced intoyeast host strain BJ2168 (from the yeast genetics stock center at theUniversity of California, Berkeley). This yeast strain has the followinggenotype: MATa, prc1-407, prb1-1122, pep4-3, leu2, trp1, ura3-52. Sincethis strain carries multiple mutant type genes of protease (prc, prb,etc), it is a desired host strain for heterologous protein expression(Zubenko, Michell, and Jones, 1980 Genetics 96: 137-146).

Yeast BJ2168 containing recombinant the plasmids is allowed to grow at30° C. with rigorous shaking in the selection medium (SD/-URA) to a logphase. The inducing agent, 3-AT (3-amino-1,2,4-triazole, Sigma #A-8056),is added to the medium to reach a concentration of 40 mM and the cultureis allowed to grow for additional 6-10 hours. After that, the culturemedium containing the secreted protein is collected by centrifugation toremove medium. Protein purification and enrichment is carried outessentially according to the standard procedure described in Rose andBroach (1990) “Propagation and expression of cloned genes in yeast: 2-μmcircle-based vectors” Math. Enzymol. 185: 234-279. Expression of scFv ismonitored by standard electrophoresis with Cormassie blue staining or bywestern blot using a tag antibody (tagging sequence such as c-Myc can beincluded in the PCR primer and integrated into the expression vector inthe PCR amplification step).

Once the condition of expression for scFv is optimized using themulti-copy plasmid vector, further optimization is done using anintegration vector pGES306. This vector differs from pGES426 only in oneaspect: it does not have the 2μ origin of replication. So it has to beintegrated into the yeast genome for stable maintenance. Experimentsshowed that the level of heterologous gene expression by GCN4 promoterdoes not rely on plasmid copy number. Mimran, Marbach, and Engelberg(2000) Biotechniques 28:552-560. Therefore, an integrated version ofGCN4 vector (present in single copy in the yeast) gives an equivalentlevel of protein expression as compared with the 2μ version plasmid(present in multiple copies usually 30-50 copies in the yeast). Theintegrated vector offers an advantage: it allows the yeast to grow inthe nutrient medium such as YPD. So yeast can grow to very high densityand the protein expression yield can be increased significantly undersuch culture condition. The secreted scFv is isolated and purified usingmethods known in the art. TABLE 1 Sequence of LoxP Sites [SEQ ID NO: 1]LoxP WT 5′-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-3′ [SEQ ID NO: 2] LoxP5115′-ATAACTTCGTATAGTATACATTATACGAAGTTAT-3′ [SEQ ID NO: 3] LoxC25′-ACAACTTCGTATAATGTATGCTATACGAAGTTAT-3′ [SEQ ID NO: 4] LoxP15′-ATAACTTCGTATAATATATGCTATACGAAGTTAT-3′ [SEQ ID NO: 5] LoxP25′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′ [SEQ ID NO: 6] LoxP35′-ATAACTTCGTATAATGTATACTATACGAAGTTAT-3′ [SEQ ID NO: 7] LoxP45′-ATAACTTCGTATAATATAAACTATACGAAGTTAT-3′ [SEQ ID NO: 8] LoxP55′-ATAACTTCGTATAATCTAACCTATACGAAGTTAT-3′ [SEQ ID NO: 9] LoxP65′-ATAACTTCGTATAACATAGCCTATACGAAGTTAT-3′ [SEQ ID NO: 10] LoxP75′-ATAACTTCGTATAACATACCCTATACGAAGTTAT-3′ [SEQ ID NO: 11] LoxP85′-ATTACCTCGTATAGCATACATTATACGAAGTTAT-3′ [SEQ ID NO: 12] LoxP95′-ATAACTTCGTATAGCATACATTATATGAAGTTAT-3′ [SEQ ID NO: 13] LoxP105′-ATTACCTCGTATAGCATACATTATATGAAGTTAT-3′

TABLE 2 Sequence of PCR primers for amplifying heavy- and light-chaingenes of human antibody. a) Heavy-chain VH 5′-primers (back primers):VH1b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQ ID NO: 14]TAC CCA TAC GAT GTT CCA GAT TAC CAG GTG CAG CTG CAG GAG TCS G-3′ VH2b:5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQ ID NO: 15] TACCCA TAC GAT GTT CCA GAT TAC CAG GTA CAG CTG CAG CAG TCA-3′ VH3b: 5′-ACCCCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQ ID NO: 16] TAC CCA TACGAT GTT CCA GAT TAC CAG GTG CAG CTA CAG CAG TGG G-3′ VH4b: 5′-ACC CCACCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQ ID NO: 17] TAC CCA TAC GATGTT CCA GAT TAC GAG GTG CAG CTG KTG GAG WCY-3′ VH5b: 5′-ACC CCA CCA AACCCA AAA AAA GAG ATC TGT ATG GCT [SEQ ID NO: 18] TAC CCA TAC GAT GTT CCAGAT TAC CAG GTC CAG CTK GTR CAG TCT GG-3′ VH6b: 5′-ACC CCA CCA AAC CCAAAA AAA GAG ATC TGT ATG GCT [SEQ ID NO: 19] TAC CCA TAC GAT GTT CCA GATTAC CAG RTC ACC TTG AAG GAG TCT G-3′ VH7b: 5′-ACC CCA CCA AAC CCA AAAAAA GAG ATC TGT ATG GCT [SEQ ID NO: 20] TAC CCA TAC GAT GTT CCA GAT TACCAG GTG CAG CTG GTG SAR TCT GG-3′ 3′-primers (forward primers): VH1f:5′-ACT GCC TCC ACC ACC GCT GCC ACC TCC GCC AGA TCC [SEQ ID NO: 21] TCCGCC GCC TGA TCC ACC ACC GCC TGA GGA GAC RGT GAC CAG GGT G-3′ VH2f:5′-ACT GCC TCC ACC ACC GCT GCC ACC TCC GCC AGA TCC [SEQ ID NO: 22] TCCGCC GCC TGA TCC ACC ACC GCC TGA GGA GAC GGT GAC CAG GGT T-3′ VH3f:5′-ACT GCC TCC ACC ACC GCT GCC ACC TCC GCC AGA TCC [SEQ ID NO: 23] TCCGCC GCC TGA TCC ACC ACC GCC TGA AGA GAC GGT GAC CAT TGT-3′ VH4f: 5′-ACTGCC TCC ACC ACC GCT GCC ACC TCC GCC AGA TCC [SEQ ID NO: 24] TCC GCC GCCTGA TCC ACC ACC GCC TGA GGA GAC GGT GAC CGT GGT CC-3′ VH5f: 5′-ACT GCCTCC ACC ACC GCT GCC ACC TCC GCC AGA TCC [SEQ ID NO: 25] TCC GCC GCC TGATCC ACC ACC GCC GGT TGG GGC GGA TGC ACT CC-3′ VH6f: 5′-ACT GCC TCC ACCACC GCT GCC ACC TCC GCC AGA TCC [SEQ ID NO: 26] TCC GCC GCC TGA TCC ACCACC GCC SGA TGG GCC CTT GGT GGA RGC-3′ b) Light-chain Vλ 5′-primers(back primers): Vλ1b: 5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA[SEQ ID NO: 27] GGT GGC AGC GGT GGT GGA GGC AGT CAG TCT GTS BTG ACG CAGCCG CC-3′ Vλ2b: 5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA [SEQID NO: 28] GGT GGC AGC GGT GGT GGA GGC AGT TCC TAT GWG CTG ACW CAG CCAC-3′ Vλ3b: 5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA [SEQ IDNO: 29] GGT GGC AGC GGT GGT GGA GGC AGT TCC TAT GAG CTG AYR CAG CYACC-3′ Vλ4b: 5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA [SEQ IDNO: 30] GGT GGC AGC GGT GGT GGA GGC AGT CAG CCT GTG CTG ACT CAR YC-3′Vλ5b: 5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGG GGA [SEQ ID NO: 31]GGT GGC AGC GGT GGT GGA GGC AGT CAG DCT GTG GTG ACY CAG GAG CC-3′ Vλ6b:5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA [SEQ ID NO: 32] GGTGGC AGC GGT GGT GGA GGC AGT CAG CCW GKG CTG ACT CAG CCM CC-3′ Vλ7b:5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA [SEQ ID NO: 33] GGTGGC AGC GGT GGT GGA GGC AGT TCC TCT GAG CTG AST CAG GAS CC-3′ Vλ8b:5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA [SEQ ID NO: 34] GGTGGC AGC GGT GGT GGA GGC AGT CAG TCT GYY CTG AYT CAG CCT-3′ Vλ9b: 5′-GGCGGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA [SEQ ID NO: 35] GGT GGC AGCGGT GGT GGA GGC AGT AAT TTT ATG CTG ACT CAG CCC C-3′ 3′-primers (forwardprimers): Vλ1f: 5′-GAG ATG GTG CAC GAT GCA CAG TTG AAG TGA ACT TGC [SEQID NO: 36] GGG GTT TTT CAG TAT CTA CGA TTC TAG GAC GGT SAS CTT GGT CC-3′Vλ2f: 5′-GAG ATG GTG CAC GAT GCA CAG TTG AAG TGA ACT TGC [SEQ ID NO: 37]GGG GTT TTT CAG TAT CTA CGA TTC GAG GAC GGT CAG CTG GGT GC-3′ c)Light-chain Vκ 5′-primers (back primers): Vκ1b: 5′-GGC GGT GGT GGA TCAGGC GGC GGA GGA TCT GGC GGA [SEQ ID NO: 38] GGT GGC AGC GGT GGT GGA GGCAGT GAC ATC CRG DTG ACC CAG TCT CC-3′ Vκ2b: 5′-GGC GGT GGT GGA TCA GGCGGC GGA GGA TCT GGC GGA [SEQ ID NO: 39] GGT GGC AGC GGT GGT GGA GGC AGTGAA ATT GTR WTG ACR CAG TCT CC-3′ Vκ3b: 5′-GGC GGT GGT GGA TCA GGC GGCGGA GGA TCT GGC GGA [SEQ ID NO: 40] GGT GGC AGC GGT GGT GGA GGC AGT GATATT GTG MTG ACB CAG WCT CC-3′ Vκ4b: 5′-GGC GGT GGT GGA TCA GGC GGC GGAGGA TCT GGC GGA [SEQ ID NO: 41] GGT GGC AGC GGT GGT GGA GGC AGT GAA ACGACA CTC ACG CAG TCT C-3′ 3′-primers (forward primers): Vκ1f: 5′-GAG ATGGTG CAC GAT GCA CAG TTG AAG TGA ACT TGC [SEQ ID NO: 42] GGG GTT TTT CAGTAT CTA CGA TTC TTT GAT TTC CAC CTT GGT CC-3′ Vκ2f: 5′-GAG ATG GTG CACGAT GCA CAG TTG AAG TGA ACT TGC [SEQ ID NO: 43] GGG GTT TTT CAG TAT CTACGA TTC TTT GAT CTC CAS CTT GGT CC-3′ Vκ3f: 5′-GAG ATG GTG CAC GAT GCACAG TTG AAG TGA ACT TGC [SEQ ID NO: 44] GGG GTT TTT CAG TAT CTA CGA TTCTTT GAT ATC CAC TTT GGT CC-3′ Vκ4f: 5′-GAG ATG GTG CAC GAT GCA CAG TTGAAG TGA ACT TGC [SEQ ID NO: 45] GGG GTT TTT CAG TAT CTA CGA TTC TTT AATCTC CAG TCG TGT CC-3′(B = C/G/T; D = A/G/T; K = G/T; M = A/C; R = A/G; S = C/G; W = A/T; andY = C/T)

TABLE 3 Sequence of oligonucleotides for modifying the cloning vectorpACT2. a) The sense strand 5′-TCGA GGC GGT GGT GGA TCA GGC [SEQ ID NO:46] GGC GGA GGA TCT GGC GGA GGT GGC AGC GGT GGT GGA GGC AGT GCG CGC TTAATT AA-3′ b) The antisense strand 5′-TCG ATT AAT TAA GCG CGC ACT GCC[SEQ ID NO: 47] TCC ACC ACC GCT GCC ACC TCC GCC AGA TCC TCC GCC GCC TGATCC ACC ACC GCC-3′

TABLE 4 Sequence of PCR primers that include loxP sites for amplifyingheavy- and light-chain genes of human antibody. a) Heavy-chain VH5′-primers (back primers): VH1b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATCTGT ATG GCT [SEQ ID NO: 14] TAC CCA TAC GAT GTT CCA GAT TAC CAG GTG CAGCTG CAG GAG TCS G-3′ VH2b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGTATG GCT [SEQ ID NO: 15] TAC CCA TAC GAT GTT CCA GAT TAC CAG GTA CAG CTGCAG CAG TCA-3′ VH3b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT[SEQ ID NO: 16] TAC CCA TAC GAT GTT CCA GAT TAC CAG GTG CAG CTA CAG CAGTGG G-3′ VH4b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQID NO: 17] TAC CCA TAC GAT GTT CCA GAT TAC GAG GTG CAG CTG KTG GAGWCY-3′ VH5b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQ IDNO: 18] TAC CCA TAC GAT GTT CCA GAT TAC CAG GTC CAG CTK GTR CAG TCTGG-3′ VH6b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQ IDNO: 19] TAC CCA TAC GAT GTT CCA GAT TAC CAG RTC ACC TTG AAG GAG TCT G-3′VH7b: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCT [SEQ ID NO: 20]TAC CCA TAC GAT GTT CCA GAT TAC CAG GTG CAG CTG GTG SAR TCT GG-3′3′-primers (forward primers): VH1′f: 5′-ACT GCC TCC ACC TGA TAA CTT CGTATA GCA TAT ATT [SEQ ID NO: 48] ATA CGA AGT TAT TGA TCC ACC ACC GCC TGAGGA GAC RGT GAC CAG GGT G-3′ VH2′f: 5′-ACT GCC TCC ACC TGA TAA CTT CGTATA GCA TAT ATT [SEQ ID NO: 49] ATA CGA AGT TAT TGA TCC ACC ACC GCC TGAGGA GAC GGT GAC CAG GGT T-3′ VH3′f: 5′-ACT GCC TCC ACC TGA TAA CTT CGTATA GCA TAT ATT [SEQ ID NO: 50] ATA CGA AGT TAT TGA TCC ACC ACC GCC TGAAGA GAC GGT GAC CAT TGT-3′ VH4′f: 5′-ACT GCC TCC ACC TGA TAA CTT CGT ATAGCA TAT ATT [SEQ ID NO: 51] ATA CGA AGT TAT TGA TCC ACC ACC GCC TGA GGAGAC GGT GAC CGT GGT CC-3′ VE5′f: 5′-ACT GCC TCC ACC TGA TAA CTT CGT ATAGCA TAT ATT [SEQ ID NO: 52] ATA CGA AGT TAT TGA TCC ACC ACC GCC GGT TGGGGC GGA TGC ACT CC-3′ VH6′f: 5′-ACT GCC TCC ACC TGA TAA CTT CGT ATA GCATAT ATT [SEQ ID NO: 53] ATA CGA AGT TAT TGA TCC ACC ACC GCC SGA TGG GCCCTT GGT GGA RGC-3′ b) Light-chain Vλ 5′-primers (back primers): Vλ1′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 54] TATACG AAG TTA TCA GGT GGA GGC AGT CAG TCT GTS BTG ACG CAG CCG CC-3′ Vλ2′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 55] TATACG AAG TTA TCA GGT GGA GGC AGT TCC TAT GWG CTG ACW CAG CCA C-3′ Vλ3′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 56] TATACG AAG TTA TCA GGT GGA GGC AGT TCC TAT GAG CTG AYR CAG CYA CC-3′ Vλ4′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 57] TATACG AAG TTA TCA GGT GGA GGC AGT CAG CCT GTG CTG ACT CAR YC-3′ Vλ5′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 58] TATACG AAG TTA TCA GGT GGA GGC AGT CAG DCT GTG GTG ACY CAG GAG CC-3′ Vλ6′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAR AAT ATA TGC [SEQ ID NO: 59] TATACG AAG TTA TCA GGT GGA GGC AGT CAG CCW GKG CTG ACT CAG CCM CC-3′ Vλ7′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 60] TATACG AAG TTA TCA GGT GGA GGC AGT TCC TCT GAG CTG AST CAG GAS CC-3′ Vλ8′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 61] TATACG AAG TTA TCA GGT GGA GGC AGT CAG TCT GYY CTG AYT CAG CCT-3′ Vλ9′b:5′-GGC GGT GGT GGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 62] TATACG AAG TTA TCA GGT GGA GGC AGT AAT TTT ATG CTG ACT CAG CCC C-3′3′-primers (forward primers): Vλ1′f: 5′-CTT CGT ATA ATG TAT GCT ATA CGAAGT TAT TAG GAC [SEQ ID NO: 63] GGT SAS CTT GGT CC-3′ Vλ2′f: 5′-CTT CGTATA ATG TAT GCT ATA CGA AGT TAT GAG GAC [SEQ ID NO: 64] GGT CAG CTG GGTGC-3′ c) Light-chain Vκ 5′-primers (back primers): Vκ1′b: 5′-GGC GGT GGTGGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 65] TAT ACG AAG TTA TCAGGT GGA GGC AGT GAC ATC CRG DTG ACC CAG TCT CC-3′ Vκ2′b: 5′-GGC GGT GGTGGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 66] TAT ACG AAG TTA TCAGGT GGA GGC AGT GAA ATT GTR WTG ACR CAG TCT CC-3′ Vκ3′b: 5′-GGC GGT GGTGGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 67] TAT ACG AAG TTA TCAGGT GGA GGC AGT GAT ATT GTG MTG ACB CAG WCT CC-3′ Vκ4′b: 5′-GGC GGT GGTGGA TCA ATA ACT TCG TAT AAT ATA TGC [SEQ ID NO: 68] TAT ACG AAG TTA TCAGGT GGA GGC AGT GAA ACG ACA CTC ACG CAG TCT C-3′ 3′-primers (forwardprimers): Vκ1′f: 5′-CTT CGT ATA ATG TAT GCT ATA CGA AGT TAT TTT GAT [SEQID NO: 69] TTC CAC CTT GGT CC-3′ Vκ2′f: 5′-CTT CGT ATA ATG TAT GCT ATACGA AGT TAT TTT GAT [SEQ ID NO: 70] CTC CAS CTT GGT CC-3′ Vκ3′f: 5′-CTTCGT ATA ATG TAT GCT ATA CGA AGT TAT TTT GAT [SEQ ID NO: 71] ATC CAC TTTGGT CC-3′ Vκ4′f: 5′-CTT CGT ATA ATG TAT GCT ATA CGA AGT TAT TTT AAT [SEQID NO: 72] CTC CAG TCG TGT CC-3′ 3′-primers (forward primers) for 2^(nd)PCR of Vλ and Vκ: Vλ/Vκf: 5′-GAG ATG GTG CAC GAT GCA CAG TTG AAG TGA ACTTGC [SEQ ID NO: 73] GGG GTT TTT CAG TAT CTA CGA TAA CTT CGT ATA ATG TATGCT- 3′(B = C/G/T; D = A/G/T; K = G/T; M = A/C; R = A/G; S = C/G; W = A/T; andY = C/T)

1. A kit, comprising: a first and second populations of haploid yeastcells of opposite mating types, the first population of haploid yeastcells comprising a library of tester expression vectors for the libraryof tester fusion proteins, each of the tester expression vectorcomprising a first transcription sequence encoding either an activationdomain or a DNA binding domain of a transcription activator, a firstnucleotide sequence encoding a first polypeptide subunit, a secondnucleotide sequence encoding a second polypeptide subunit, and a linkersequence encoding a linker peptide that links the first nucleotidesequence and the second nucleotide sequence, wherein the firstpolypeptide subunit, the linker peptide, the second polypeptide areexpressed as a tester fusion protein with either the activation domainor the DNA binding domain of the transcription activator; the secondpopulation of haploid yeast cells comprises a target expression vectorfor a target fusion protein, the target expression vector comprising asecond transcription sequence encoding either the activation domain orthe DNA binding domain of the transcription activator which is notexpressed by the library of tester expression vectors, and a targetsequence encoding the target protein or peptide, wherein the targetprotein or peptide is expressed as a fusion protein with either theactivation domain or the DNA binding domain of the transcriptionactivator which is not expressed by the library of tester expressionvectors, and either the first or second population of haploid yeastcells comprises a reporter construct comprising a reporter gene whoseexpression is under transcriptional control of the transcriptionactivator.
 2. The kit of claim 1, wherein the second population ofhaploid yeast cells comprises a plurality of target expression vectors,each of the target expression vectors encoding a second transcriptionsequence encoding either the activation domain or the DNA binding domainof the transcription activator which is not expressed by the library oftester expression vectors; and a target sequence encoding the targetprotein or peptide.
 3. The kit of claim 1, wherein the haploid yeastcells of opposite mating types are α and a type strains of yeast.
 4. Thekit of claim 1, wherein the first polypeptide subunit comprises anantibody heavy-chain variable region, and the second polypeptide subunitcomprises an antibody light-chain variable region.
 5. The method ofclaim 1, wherein the protein encoded by the reporter gene is selectedfrom the group consisting of β-galactosidase, α-galactosidase,luciferase, β-glucuronidase, chloramphenicol acetyl transferase,secreted embryonic alkaline phosphatase, green fluorescent protein,enhanced blue fluorescent protein, enhanced yellow fluorescent protein,and enhanced cyan fluorescent protein.
 6. The kit of claim 1, whereinthe diversity of the tester fusion proteins encoded by the library oftester expression vectors is at least 1×10⁶.
 7. The kit of claim 1,wherein the diversity of the tester fusion proteins encoded by thelibrary of tester expression vectors is at least 1×10⁷.
 8. The kit ofclaim 1, wherein the diversity of the tester fusion proteins encoded bythe library of tester expression vectors is at least 1×10⁸.
 9. The kitof claim 1, wherein the target fusion protein comprises an antigenassociated with a disease state.
 10. The kit of claim 1, wherein thetarget fusion protein comprises a tumor-surface antigen.
 11. The kit ofclaim 1, wherein the target fusion protein comprises a human growthfactor selected from the group consisting of epidermal growth factors,transferrin, insulin-like growth factor, transforming growth factors,interleukin-1, and interleukin-2.
 12. The kit of claim 1, wherein thefirst polypeptide subunit and the second polypeptide subunit are encodedby variable regions of immunoglobulin genes of a human, non-humanprimates, or rodent.
 13. The kit of claim 1, wherein the firstpolypeptide subunit and the second polypeptide subunit are encodedrespectively by a heavy-chain variable region and a light-chain variableregion of a human immunoglobulin gene.
 14. The kit of claim 1, whereinthe first polypeptide subunit is encoded by a heavy-chain variableregion of a first human immunoglobulin gene, and the second polypeptidesubunit is encoded by a light chain variable region of a second humanimmunoglobulin gene different from the first human immunoglobulin gene.15. A kit, comprising: a first and second populations of haploid yeastcells of opposite mating types, the first population of haploid yeastcells comprising a library of tester expression vectors for the libraryof tester fusion proteins, each of the tester expression vectorcomprising a first transcription sequence encoding either an activationdomain or a DNA binding domain of a transcription activator, a firstnucleotide sequence encoding a first polypeptide subunit, a secondnucleotide sequence encoding a second polypeptide subunit, and a linkersequence encoding a linker peptide that links the first nucleotidesequence and the second nucleotide sequence, wherein the firstpolypeptide subunit, the linker peptide, the second polypeptide areexpressed as a tester fusion protein with either the activation domainor the DNA binding domain of the transcription activator, wherein eitherthe first or second population of haploid yeast cells comprises areporter construct comprising a reporter gene whose expression is undertranscriptional control of the transcription activator.
 16. The kit ofclaim 15, wherein the second population of haploid yeast cells comprisesa target expression vector for a target fusion protein, the targetexpression vector comprising a second transcription sequence encodingeither the activation domain or the DNA binding domain of thetranscription activator which is not expressed by the library of testerexpression vectors.
 17. The kit of claim 15, wherein the diversity ofthe tester fusion proteins encoded by the library of tester expressionvectors is at least 1×10⁶.
 18. The kit of claim 15, wherein thediversity of the tester fusion proteins encoded by the library of testerexpression vectors is at least 1×10⁷.
 19. The kit of claim 15, whereinthe diversity of the tester fusion proteins encoded by the library oftester expression vectors is at least 1×10⁸.
 20. The kit of claim 15,wherein the first polypeptide subunit comprises an antibody heavy-chainvariable region, and the second polypeptide subunit comprises anantibody light-chain variable region.
 21. The kit of claim 15, whereinthe first polypeptide subunit and the second polypeptide subunit areencoded by variable regions of immunoglobulin genes of a human,non-human primates, or rodent.
 22. The kit of claim 15, wherein thefirst polypeptide subunit and the second polypeptide subunit are encodedrespectively by a heavy-chain variable region and a light-chain variableregion of a human immunoglobulin gene.
 23. The kit of claim 15, whereinthe first polypeptide subunit is encoded by a heavy-chain variableregion of a first human immunoglobulin gene, and the second polypeptidesubunit is encoded by a light chain variable region of a second humanimmunoglobulin gene different from the first human immunoglobulin gene.24. The kit of claim 15, wherein the haploid yeast cells of oppositemating types are α and a type strains of yeast.
 25. The kit of claim 15,wherein the transcription activator is a transcription activator havingseparable DNA-binding and transcription activation domains.
 26. The kitof claim 15, wherein the transcription activator is selected from thegroup consisting of GAL4, GCN4, and ADR1 transcription activator.
 27. Amethod for generating a library of yeast expression vectors, comprising:a) transforming into yeast cells i) a linearized yeast expression vectorhaving a 5′- and 3′-terminus sequence at a first site of linearization;and ii) a library of first insert nucleotide sequences that are linear,double stranded, each of the first insert sequences comprising a firstnucleotide sequence encoding a first polypeptide subunit, a 5′- and3′-flanking sequence at the ends of the first insert sequence which aresufficiently homologous to the 5′- and 3′-terminus sequences of thevector at the first site of linearization, respectively, to enablehomologous recombination to occur; b) having homologous recombinationoccur between the linearized yeast expression vector and the firstinsert sequence in the transformed yeast cells, such that the firstinsert sequence is included in the expression vector; c) isolating fromthe transformed yeast cells the expression vectors that contain thelibrary of the first insert sequences; d) linearizing the expressionvectors containing the library of the first insert sequences to generatea 5′- and 3′-terminus sequence at a second site of linearization; e)transforming into yeast cells i) the linearized expression vectors instep d), and ii) a library of second insert nucleotide sequences thatare linear, double stranded, each of the second insert sequencescomprising a second nucleotide sequence encoding a second polypeptidesubunit, a 5′- and 3′-flanking sequence at the ends of the second insertsequence which are sufficiently homologous to the 5′- and 3′-terminussequences of the vector at the second site of linearization,respectively, to enable homologous recombination to occur; and f) havinghomologous recombination occur between the linearized yeast expressionvector at the second linearization site and the second insert sequencein the transformed yeast cells, such that the second insert sequence isincluded in the vector and the first and second nucleotide sequences arelinked by a linker sequence; wherein the expression vector expresses thefirst polypeptide subunit, the second polypeptide subunit, and thelinker polypeptide as a single fusion protein in the transformed yeastcells by the library of yeast expression vectors; and the first andsecond nucleotide sequences each independently varies within the libraryof expression vectors.
 28. The method of claim 27, wherein the 5′- or3′-flanking sequence of the first or the second insert nucleotidesequence is between 30-120 bp in length.
 29. The method of claim 27,wherein the 5′- or 3′-flanking sequence of the first or the secondinsert nucleotide sequence is between 40-90 bp in length.
 30. The methodof claim 27, wherein the 5′- or 3′-flanking sequence of the first or thesecond insert nucleotide sequence is between 60-80 bp in length.
 31. Themethod of claim 27, wherein the linker sequence is between 30-120 bp inlength.
 32. The method of claim 27, wherein the linker sequence isbetween 45-102 bp in length.
 33. The method of claim 27, wherein thelinker sequence is between 45-63 bp in length.
 34. The method of claim27, wherein the linker sequence comprises a nucleotide sequence encodingan amino acid sequence of Gly-Gly-Gly-Gly-Ser in 3 or 4 tandem repeats.35. The method of claim 27, wherein the 5′- and 3′-flanking sequences atthe ends of the first or second insert nucleotide sequence comprise a5′- and 3′-site-specific recombination site, respectively, that arerecognized by a site-specific recombinase.
 36. The method of claim 35,wherein one of the 5′- and 3′-site-specific recombination sites iscoliphase P1 loxP, and the other is a mutant loxP sequence.
 37. Themethod of claim 35, wherein the 5′- and 3′-site-specific recombinationsites are each independently selected from the group consisting of SEQID Nos 1-13.
 38. The method of claim 35, wherein the site-specificrecombinase is CRE recombinase.
 39. The method of claim 35, furthercomprising: causing site-specific recombination between the members ofthe library of the yeast expression vectors at the 5′- and3′-recombination sites, the recombination resulting in exchange of thefirst or second nucleotide sequences between the members of the libraryof the yeast expression vectors.
 40. The method of claim 39, wherein therecombination is caused by expression of a recombinase that is induciblycontrolled in the yeast cells.
 41. The method of claim 40, wherein the5′- and 3′-recombination sites are different loxP sequences, and therecombination is caused by inducible expression of CRE recombinase inthe yeast cells.
 42. The method of claim 27, wherein the diversity ofthe first or second polypeptide subunit encoded by the library of yeastexpression vectors is at least 10³.
 43. The method of claim 27, whereinthe diversity of the first or second polypeptide subunit encoded by thelibrary of yeast expression vectors is at least 10³.
 44. The method ofclaim 27, wherein the diversity of the first or second polypeptidesubunit encoded by the library of yeast expression vectors is at least10⁵.
 45. The method of claim 27, wherein the diversity of the fusionproteins encoded by the library of yeast expression vectors is at least1×10⁸.
 46. The method of claim 27, wherein the diversity of the fusionproteins encoded by the library of yeast expression vectors is at least1×10¹⁰.
 47. The method of claim 27, wherein the diversity of the fusionproteins encoded by the library of yeast expression vectors is at least1×10¹².
 48. The method of claim 27, wherein each of the expressionvectors further comprises a sequence encoding an affinity tag.
 49. Themethod of claim 48, wherein the affinity tag is selected from the groupconsisting of a polyhistidine tag, polyarginine tag,glutathione-S-transferase, maltose binding protein, staphylococcalprotein A tag, and an EE-epitope tag.
 50. The method of claim 48,wherein the first nucleotide sequence and the second nucleotide sequencerespectively encode a heavy chain variable region and a light chainvariable region of immunoglobulin genes of a human, non-human primates,or rodent.
 51. The method of claim 48, wherein the first nucleotidesequence and the second nucleotide sequence respectively encode a heavychain variable region and a light chain variable region of a humanimmunoglobulin gene.
 52. The method of claim 48, the linearized yeastexpression vector further comprising: a transcription sequence encodingan activation domain or a DNA binding domain of a transcriptionactivator.
 53. The method of claim 52, wherein the transcriptionsequence is capable of being expressed as a fusion protein with thesingle fusion protein comprising the antibody heavy chain variableregion, the antibody light chain variable region, and the linkerpolypeptide.
 54. The method of claim 52, wherein the transcriptionactivator is a transcription activator having separable DNA-binding andtranscription activation domains.
 55. The method of claim 52, whereinthe transcription activator is selected from the group consisting ofGAL4, GCN4, and ADR1 transcription activator.